Synthetic triterpenoids and methods of use in the treatment of disease

ABSTRACT

The present invention concerns methods for treating and preventing renal/kidney disease, insulin resistance/diabetes, fatty liver disease, and/or endothelial dysfunction/cardiovascular disease using synthetic triterpenoids, optionally in combination with a second treatment or prophylaxis.

The present application claims the benefit of priority to U.S.Provisional Application Nos. 61/020,624, filed Jan. 11, 2008, and61/109,114, filed Oct. 28, 2008, the entire contents of each of whichare incorporated by reference herein.

BACKGROUND OF THE INVENTION

I. Field of the Invention

The present invention relates generally to the fields of biology andmedicine. More particularly, it concerns compositions and methods fortreating and/or preventing renal/kidney disease (RKD), insulinresistance, diabetes, endothelial dysfunction, fatty liver disease, andcardiovascular disease (CVD).

II. Description of Related Art

Renal failure, resulting in inadequate clearance of metabolic wasteproducts from the blood and abnormal concentrations of electrolytes inthe blood, is a significant medical problem throughout the world,especially in developed countries. Diabetes and hypertension are amongthe most important causes of chronic renal failure, also known aschronic kidney disease (CKD), but it is also associated with otherconditions such as lupus or systemic cardiovascular disease. Dysfunctionof the vascular endothelium commonly occurs in such conditions and isbelieved to be a major contributing factor in the development of chronickidney disease. Acute renal failure may arise from exposure to certaindrugs (e.g., acetaminophen) or toxic chemicals or fromischemia-reperfusion injury associated with shock or surgical proceduressuch as transplantation, and may ultimately result in CKD. In manypatients, CKD advances to end-stage renal disease (ESRD) in which thepatient requires kidney transplantation or regular dialysis to continueliving. Both of these procedures are highly invasive and associated withsignificant side effects and quality of life issues. Although there areeffective treatments for some complications of renal failure, such ashyperparathyroidism and hyperphosphatemia, no available treatment hasbeen shown to halt or reverse the underlying progression of renalfailure. Thus, agents that can improve compromised renal function wouldrepresent a significant advance in the treatment of renal failure.

Triterpenoids, biosynthesized in plants by the cyclization of squalene,are used for medicinal purposes in many Asian countries; and some, likeursolic and oleanolic acids, are known to be anti-inflammatory andanti-carcinogenic (Huang et al., 1994; Nishino et al., 1988). However,the biological activity of these naturally-occurring molecules isrelatively weak, and therefore the synthesis of new analogs to enhancetheir potency was undertaken (Honda et al., 1997; Honda et al., 1998).An ongoing effort for the improvement of anti-inflammatory andantiproliferative activity of oleanolic and ursolic acid analogs led tothe discovery of 2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid(CDDO) and related compounds (Honda et al., 1997, 1998, 1999, 2000a,2000b, 2002; Suh et al., 1998; 1999; 2003; Place et al., 2003; Liby etal., 2005). Several potent derivatives of oleanolic acid wereidentified, including methyl-2-cyano-3,12-dioxooleana-1,9-dien-28-oicacid (CDDO-Me; RTA 402). RTA 402 suppresses the induction of severalimportant inflammatory mediators, such as iNOS, COX-2, TNFα, and IFNγ,in activated macrophages. RTA 402 has also been reported to activate theKeap1/Nrf2/ARE signaling pathway resulting in the production of severalanti-inflammatory and antioxidant proteins, such as heme oxygenase-1(HO-1). These properties have made RTA 402 a candidate for the treatmentof neoplastic and proliferative diseases, such as cancer. The ability ofthis compound and related molecules to treat and/or prevent kidneydisease and cardiovascular disease remains untested.

SUMMARY OF THE INVENTION

The present invention provides new methods for treating and/orpreventing renal/kidney disease (RKD), insulin resistance, diabetes,endothelial dysfunction, fatty liver disease, cardiovascular disease(CVD), and related disorders. Compounds covered by the generic orspecific formulas below or specifically named are referred to as“compounds of the invention,” “compounds of the present invention,” or“synthetic triterpenoids” in the present application.

In one aspect of the present prevention, methods are provided fortreating or preventing renal/kidney disease (RKD), insulin resistance,diabetes, endothelial dysfunction, fatty liver disease, orcardiovascular disease (CVD) in a subject comprising, administering tosaid subject a pharmaceutically effective amount of a compound havingthe structure:

wherein R₁ is: —CN, or C₁-C₁₅-acyl or C₁-C₁₅-alkyl, wherein either ofthese groups is heteroatom-substituted or heteroatom-unsubstituted; or apharmaceutically acceptable salt, hydrate or solvate thereof.

In some embodiments, methods are provided for treating RKD. In somevariations, the RKD is diabetic nephropathy (DN). In other variations,the RKD results from a toxic insult, for example, wherein the toxicinsult results from an imaging agent or a drug. For example, the drugmay be a chemotherapeutic agent. In a further variation, the RKD resultsfrom ischemia/reperfusion injury. In yet a further variation, the RKDresults from diabetes or hypertension. In still further variations, theRKD results from an autoimmune disease. In other variations, the RKD ischronic RKD. In still other variations, the RKD is acute RKD.

In some embodiments, the subject has undergone or is undergoingdialysis. In some embodiments, the subject has undergone or is acandidate to undergo kidney transplant. In some embodiments, the subjecthas RKD and insulin resistance. In some variations on the aboveembodiments, the subject has RKD, insulin resistance and endothelialdysfunction. In some embodiments, the subject has RKD and diabetes. Insome embodiments, the subject has insulin resistance.

In some embodiments, the subject has diabetes. The pharmaceuticallyeffective amount of the compound may also effectively treat one or morecomplications associated with diabetes. For example, the complicationscan be selected from the group consisting of obesity, hypertension,atherosclerosis, coronary heart disease, stroke, peripheral vasculardisease, hypertension, nephropathy, neuropathy, myonecrosis, diabeticfoot ulcers and other diabetic ulcers, retinopathy and metabolicsyndrome (syndrome X). Also, for example, the complication can bemetabolic syndrome (syndrome X). In some variations, the diabetesresults from insulin resistance.

In some embodiments, the subject has RKD and endothelial dysfunction. Inother embodiments, the subject has RKD and cardiovascular disease. Insome embodiments, the subject has CVD. In some variations, the CVDresults from endothelial dysfunction.

In some embodiments, the subject has endothelial dysfunction and/orinsulin resistance. In some embodiments, the subject has fatty liverdisease. In some variations, the fatty liver disease is non-alcoholicfatty liver disease. In other variations, the fatty liver disease isalcoholic fatty liver disease. In some variations, the subject has fattyliver disease and one or more of the following disorders: renal/kidneydisease (RKD), insulin resistance, diabetes, endothelial dysfunction,and cardiovascular disease (CVD).

In some embodiments, the methods further comprise identifying a subjectin need of treatment of any of the diseases, dysfunctions, resistancesor disorders listed herein. In some embodiments, the subject has afamily or patient history of any of the diseases, dysfunctions,resistances or disorders listed herein. In some embodiments, the subjectexhibits symptoms of any of the diseases, dysfunctions, resistances ordisorders listed herein.

In another aspect of the invention, a method is provided for improvingglomerular filtration rate or creatinine clearance in a subjectcomprising, administering to said subject a pharmaceutically effectiveamount of a compound having the structure of Formula I, or apharmaceutically acceptable salt, hydrate or solvate thereof.

In some embodiments, the compound is administered locally. In someembodiments, the compound is administered systemically. In someembodiments, the compound is administered orally, intraadiposally,intraarterially, intraarticularly, intracranially, intradermally,intralesionally, intramuscularly, intranasally, intraocularally,intrapericardially, intraperitoneally, intrapleurally,intraprostatically, intrarectally, intrathecally, intratracheally,intratumorally, intraumbilically, intravaginally, intravenously,intravesicularlly, intravitreally, liposomally, locally, mucosally,orally, parenterally, rectally, subconjunctivally, subcutaneously,sublingually, topically, transbuccally, transdermally, vaginally, incremes, in lipid compositions, via a catheter, via a lavage, viacontinuous infusion, via infusion, via inhalation, via injection, vialocal delivery, via localized perfusion, bathing target cells directly,or any combination thereof. For example, in some variations, thecompound is administered intravenously, intra-arterially or orally. Forexample, in some variations, the compound is administered orally.

In some embodiments, the compound is formulated as a hard or softcapsule, a tablet, a syrup, a suspension, a solid dispersion, a wafer,or an elixir. In some variations, the soft capsule is a gelatin capsule.In variations, the compound is formulated as a solid dispersion. In somevariations the hard capsule, soft capsule, tablet or wafer furthercomprises a protective coating. In some variations, the formulatedcompound comprises an agent that delays absorption. In some variations,the formulated compound further comprises an agent that enhancessolubility or dispersibility. In some variations, the compound isdispersed in a liposome, an oil in water emulsion or a water in oilemulsion.

In some embodiments, the pharmaceutically effective amount is a dailydose from about 0.1 mg to about 500 mg of the compound. In somevariations, the daily dose is from about 1 mg to about 300 mg of thecompound. In some variations, the daily dose is from about 10 mg toabout 200 mg of the compound. In some variations, the daily dose isabout 25 mg of the compound. In other variations, the daily dose isabout 75 mg of the compound. In still other variations, the daily doseis about 150 mg of the compound. In further variations, the daily doseis from about 0.1 mg to about 30 mg of the compound. In some variations,the daily dose is from about 0.5 mg to about 20 mg of the compound. Insome variations, the daily dose is from about 1 mg to about 15 mg of thecompound. In some variations, the daily dose is from about 1 mg to about10 mg of the compound. In some variations, the daily dose is from about1 mg to about 5 mg of the compound.

In some embodiments, the pharmaceutically effective amount is a dailydose is 0.01-25 mg of compound per kg of body weight. In somevariations, the daily dose is 0.05-20 mg of compound per kg of bodyweight. In some variations, the daily dose is 0.1-10 mg of compound perkg of body weight. In some variations, the daily dose is 0.1-5 mg ofcompound per kg of body weight. In some variations, the daily dose is0.1-2.5 mg of compound per kg of body weight.

In some embodiments, the pharmaceutically effective amount isadministered in a single dose per day. In some embodiments, thepharmaceutically effective amount is administered in two or more dosesper day.

In some embodiments, the treatment method further comprises a secondtherapy. In some variations, the second therapy comprises administeringto said subject a pharmaceutically effective amount of a second drug. Insome embodiments, the second drug is a cholesterol lowering drug, ananti-hyperlipidemic, a calcium channel blocker, an anti-hypertensive, oran HMG-CoA reductase inhibitor. Non-limiting examples of second drugsare amlodipine, aspirin, ezetimibe, felodipine, lacidipine,lercanidipine, nicardipine, nifedipine, nimodipine, nisoldipine andnitrendipine. Further non-limiting examples of second drugs areatenolol, bucindolol, carvedilol, clonidine, doxazosin, indoramin,labetalol, methyldopa, metoprolol, nadolol, oxprenolol,phenoxybenzamine, phentolamine, pindolol, prazosin, propranolol,terazosin, timolol and tolazoline. In some variations, the second drugis a statin. Non-limiting examples of statins are atorvastatin,cerivastatin, fluvastatin, lovastatin, mevastatin, pitavastatin,pravastatin, rosuvastatin and simvastatin. In some variations, thesecond drug is a dipeptidyl peptidase-4 (DPP-4) inhibitor. Non-limitingexamples of DPP-4 inhibitors are sitagliptin, vildagliptin, SYR-322, BMS477118 and GSK 823093. In some variations, the second drug is abiguanide. For example, the biguanide can be metformin. In somevariations, the second drug is a thiazolidinedione (TZD). Non-limitingexamples of TZDs are pioglitazone, rosiglitazone and troglitazone. Insome variations, the second drug is a sulfonylurea derivative.Non-limiting examples of sulfonyl urea derivatives are tolbutamide,acetohexamide, tolazamide, chlorpropamide, glipizide, glyburide,glimepiride and gliclazide. In some variations, the second drug is ameglitinide. Non-limiting examples of meglitinides include repaglinide,mitiglinide and nateglinide. In some variations, the second drug isinsulin. In some variations, the second drug is an alpha-glucosidaseinhibitor. Non-limiting examples of alpha-glucosidase inhibitors areacarbose, miglitol and voglibose. In some variations, the second drug isa glucagon-like peptide-1 analog. Non-limiting examples of glucagon-likepeptide-1 analogs are exenatide and liraglutide. In some variations, thesecond drug is a gastric inhibitory peptide analog. In some variations,the second drug is a GPR40 agonist. In some variations, the second drugis a GPR119 agonist. In some variations the second drug is a GPR30agonist. In some variations, the second drug is a glucokinase activator.In some variations, the second drug is a glucagon receptor antagonist.In some variations, the second drug is an amylin analog. A non-limitingexample of an amylin analog is pramlintide. In some variations, thesecond drug is an IL-1β receptor antagonist. A non-limiting examples ofa IL-1β receptor antagonist is anakinra. In some variations, the seconddrug is an endocannabinoid receptor antagonist or inverse agonist. Anon-limiting example of a endocannabinoid receptor antagonist or inverseagonist is rimonabant. In some variations, the second drug is Orlistat.In some variations, the second drug is Sibutramine. In some variations,the second drug is a growth factor. Non-limiting examples of growthfactors are TGF-β1, TGF-β2, TGF-β1.2, VEGF, insulin-like growth factor Ior II, BMP2, BMP4, BMP7, a GLP-1 analog, a GIP analog, a DPP-IVinhibitor, a GPR119 agonist, a GPR40 agonist, gastrin, EGF,betacellulin, KGF, NGF, insulin, growth hormone, HGF, an FGF, an FGFhomologue, PDGF, Leptin, prolactin, placental lactogen, PTHrP, activin,inhibin, and INGAP. Further non-limiting examples of growth factors areparathyroid hormone, calcitonin, interleukin-6, and interleukin-11.

In some embodiments, the subject is a primate. In some variations, theprimate is a human. In other variations, the subject is a cow, horse,dog, cat, pig, mouse, rat or guinea pig.

In some embodiments, the compound is defined as:

wherein Y is: —H, hydroxy, amino, halo, or C₁-C₁₄-alkoxy,C₂-C₁₄-alkenyloxy, C₂-C₁₄-alkynyloxy, C₁-C₁₄-aryloxy, C₂-C₁₄-aralkoxy,C₁-C₁₄-alkylamino, C₂-C₁₄-alkenylamino, C₂-C₁₄-alkynylamino,C₁-C₁₄-arylamino, C₃-C₁₀-aryl, or C₂-C₁₄-aralkylamino, wherein any ofthese groups is heteroatom-substituted or heteroatom-unsubstituted; or apharmaceutically acceptable salt, hydrate or solvate thereof.

In some embodiments, Y is a heteroatom-unsubstituted C₁-C₄-alkylamino,such that the compound of the invention is, for example:

In some embodiments, Y is a heteroatom-substituted orheteroatom-unsubstituted C₂-C₄-alkylamino, such that the compound of theinvention is, for example:

In some embodiments, Y is a heteroatom-substituted orheteroatom-unsubstituted C₁-C₄-alkoxy, such as aheteroatom-unsubstituted C₁-C₂-alkoxy. For example, one non-limitingexample of such a compound is:

In some embodiments, at least a portion of the CDDO-Me is present as apolymorphic form, wherein the polymorphic form is a crystalline formhaving an X-ray diffraction pattern (CuKα) comprising significantdiffraction peaks at about 8.8, 12.9, 13.4, 14.2 and 17.4° 2θ. Innon-limiting examples, the X-ray diffraction pattern (CuKα) issubstantially as shown in FIG. 12A or FIG. 12B. In other variations, atleast a portion of the CDDO-Me is present as a polymorphic form, whereinthe polymorphic form is an amorphous form having an X-ray diffractionpattern (CuKα) with a halo peak at approximately 13.5° 2θ, substantiallyas shown in FIG. 12C, and a T_(g). In some variations, the compound isan amorphous form. In some variations, the compound is a glassy solidform of CDDO-Me, having an X-ray powder diffraction pattern with a halopeak at about 13.5° 2θ, as shown in FIG. 12C, and a T_(g). In somevariations, the T_(g) value falls within a range of about 120° C. toabout 135° C. In some variations, the T_(g) value is from about 125° C.to about 130° C.

In some embodiments, Y is hydroxy, such that the compound of theinvention is, for example:

In some embodiments, the compound is:

In some embodiments, the compound is defined as:

wherein Y′ is a heteroatom-substituted or heteroatom-unsubstitutedC₁-C₁₄-aryl; or a pharmaceutically acceptable salt, hydrate or solvatethereof.

In some embodiments, the compound is:

In some variations of the above methods, the compound is substantiallyfree from optical isomers thereof. In some variations of the abovemethods, the compound is in the form of a pharmaceutically acceptablesalt. In other variations of the above methods, the compound is not asalt.

In some embodiments, the compound is formulated as a pharmaceuticalcomposition comprising (i) a therapeutically effective amount of thecompound and (ii) an excipient is selected from the group consisting of(A) a carbohydrate, carbohydrate derivative, or carbohydrate polymer,(B) a synthetic organic polymer, (C) an organic acid salt, (D) aprotein, polypeptide, or peptide, and (E) a high molecular weightpolysaccharide. In some variations, the excipient is a synthetic organicpolymer. In some variations, the excipient is selected from the groupconsisting of a hydroxpropyl methyl cellulose, apoly[1-(2-oxo-1-pyrrolidinyl)ethylene or copolymer thereof, and amethacrylic acid-methylmethacrylate copolymer. In some variations, theexcipient is hydroxpropyl methyl cellulose phthalate ester. In somevariations, the excipient is PVP/VA. In some variations, the excipientis a methacrylic acid-ethyl acrylate:copolymer (1:1). In somevariations, the excipient is copovidone.

Any embodiment discussed herein with respect to one aspect of theinvention applies to other aspects of the invention as well, unlessspecifically noted.

Other objects, features and advantages of the present invention willbecome apparent from the following detailed description and anyaccompanying drawings. It should be understood, however, that thedetailed description and any specific examples or drawings provided,while indicating specific embodiments of the invention, are given by wayof illustration only, since various changes and modifications within thespirit and scope of the invention will become apparent to those skilledin the art from this detailed description.

BRIEF DESCRIPTION OF THE DRAWINGS

The following drawings form part of the present specification and areincluded to further demonstrate certain aspects of the presentinvention. The invention may be better understood by reference to one ormore of these drawings in combination with the detailed description ofspecific embodiments presented herein.

FIGS. 1 a-d—RTA 402 reduces renal damage following ischemia-reperfusion.Mice were administered RTA 402 at 2 mg/kg or simply the vehicle (sesameoil) daily by oral gavage beginning on Day 2. On Day 0, a clamp wasplaced on the left renal artery for 17 minutes and then removed toinduce ischemia-reperfusion. (FIG. 1 a) On Day 1, blood was collectedfrom animals that were subjected to clamping and “sham” control animalsthat underwent surgery without clamping of the renal artery. Blood ureanitrogen (BUN) levels were measured as a surrogate for renal damage.(FIGS. 1 b-d) Sections of kidneys from RTA 402-treated orvehicle-treated mice were scored for histological damage (FIGS. 1 b & 1d) and inflammation (FIG. 1 c). (FIG. 1 d) Black arrows (vehicle group)show two of many severely damaged tubules in the outer medulla. Redarrows (RTA 402 group) show two of many undamaged tubules in the outermedulla.

FIGS. 2 a-c—RTA 402 reduces cisplatin-induced renal toxicity. Rats wereadministered RTA 402 at 10 mg/kg or simply the vehicle (sesame oil)every day by oral gavage beginning on Day −1. On Day 0, the ratsreceived an intravenous injection of cisplatin at 6 mg/kg. Blood sampleswere drawn on the indicated days and the levels of creatinine (FIG. 2 a)and blood urea nitrogen (BUN) (FIG. 2 b) were measured as markers ofrenal damage. A statistically significant difference was observedbetween the vehicle-treated and RTA 402-treated groups on Day 3(creatinine) and Day 5 (creatinine and BUN). (FIG. 2 c) Less damage tothe proximal tubules is observed in RTA 402-treated animals compared tovehicle-treated animals.

FIGS. 3 a-d—RTA 402 reduces serum creatinine levels in monkeys, dogs,and rats. (FIG. 3 a) Cynomolgus monkeys were administered RTA 402 orallyat the indicated doses once daily for 28 days. The percent reduction ofserum creatinine on Day 28 in RTA 402-treated monkeys relative tovehicle-treated control monkeys is shown. (FIG. 3 b) RTA 402 wasadministered orally to beagle dogs at the indicated doses daily forthree months. Control animals received vehicle (sesame oil). The percentchange in serum creatinine at the three-month time point relative tobaseline is shown. (FIG. 3 c) Sprague-Dawley rats were administered RTA402 orally at the indicated doses once daily for a period of one month.The percent reduction of serum creatinine at study completion in RTA402-treated rats relative to vehicle-treated control rats is shown.(FIG. 3 d) Sprague-Dawley rats were administered the amorphous form ofRTA 402 orally at the indicated doses once daily for a period of threemonths. The percent reduction of serum creatinine at study completion inRTA 402-treated rats relative to vehicle-treated control rats is shown.Note: in FIGS. 3A, 3C and 3D, “% reduction” on the vertical axisindicates percent change. For example, a reading of −15 on this axisindicates a 15% reduction in serum creatinine.

FIGS. 4A-B—RTA 402 reduces serum creatinine levels and increases theestimated glomerular filtration rate (eGFR) in human patients withcancer. FIG. 4A: Serum creatinine was measured in RTA 402-treatedpatients enrolled in a Phase I clinical trial for the treatment ofcancer. The patients were administered RTA 402 (p.o.) once daily for 21days at doses ranging from 5 to 1,300 mg/day. The percent reduction ofserum creatinine relative to baseline levels is shown for the indicatedstudy days. Significant decreases in serum creatinine levels wereobserved on Days 15 and 21. FIG. 4B: The estimated glomerular filtrationrate (eGFR) was calculated for the patients in FIG. 4A. Significantimprovements in the eGFR were observed in both groups. All patients:n=24; patients with baseline≧1.5: n=5. For FIGS. 4A and 4B, * indicatesp≧0.04; † indicates p=0.01, and ‡ indicates p<0.01. Note: in FIG. 4A, “%Reduction from Baseline” on the vertical axis indicates percent change.For example, a reading of −15 on this axis indicates a 15% reduction inserum creatinine.

FIG. 5—RTA 402 increases GFR in human patients with cancer. Estimatedglomerular filtration rate (eGFR) was measured in RTA 402-treatedpatients enrolled in a multi-month clinical trial for the treatment ofcancer. All patients (n=11) dosed through six months were included inthe analysis. The dosing information for these patients is provided inExample 5, below.

FIG. 6-RTA 402 Activity Correlates with Severity. Reduction ofhemoglobin Alc is presented as a fraction of the initial baseline value.Groups with higher baselines, e.g., mean baseline≧7.0% Alc or ≧7.6% Alc,showed greater reduction. The intent-to-treat (ITT) group includes allpatients (n=53), including those starting at a normal Alc value.

FIG. 7—RTA 402 Activity is Dose Dependent. Reduction of hemoglobin Alcis presented relative to the initial baseline value. The bar graph showsmean results for all patients, all patients with baseline Alcvalues≧7.0%, individual dose cohorts from the ≧7.0% group, and patientswith Stage 4 renal disease (GFR 15-29 mL/min), wherein n is the numberof patients in each group.

FIG. 8—RTA 402 Reduces Circulating Endothelial Cells (CECs) andiNOS-positive CECs. The change in the mean number of CECs in cells/mL isshown for intent-to-treat (ITT) and elevated baseline groups, bothbefore and after the 28 day RTA treatment. The reduction for theIntent-to-treat group was approximately 20%, and the reduction in theelevated baseline group (>5 CECs/ml) was approximately 33%. The fractionof iNOS-positive CECs was reduced approximately 29%.

FIG. 9—Reversible Dose Dependent GFR Increase in 28 Days. Treatment withRTA 402 increases GFR dose-dependently. All evaluable patients wereincluded. An improvement of >30% was noted in patients with Stage 4renal disease.

FIGS. 10A-B—Reduction of Markers of Diabetic Nephropathy Severity andOutcome. Improvements in Adiponectin (FIG. 10A) and Angiotensin II (FIG.10B), which are elevated in diabetic nephropathy (DN) patients andcorrelate with renal disease severity. Adiponectin predicts all-causemortality and end stage renal disease in DN patients. All available dataincluded.

FIGS. 11A-C—RTA 402 Significantly Reduces Uremic Solutes. The graphspresent mean changes in BUN (FIG. 11A), phosphorus (FIG. 11B), and uricacid (FIG. 11C) for all patients and for those patients showing elevatedbaseline values of a particular solute.

FIGS. 12A-C—X-ray Powder Diffraction (XRPD) Spectra of Forms A and B ofRTA 402. FIG. 12A shows unmicronized Form A; FIG. 12B shows micronizedForm A; FIG. 12C shows Form B.

FIG. 13—Modulated Differential Scanning Calorimetry (MDSC) Curve of FormA RTA 402. The section of the curve shown in the expanded view isconsistent with a glass transition temperature (T_(g)).

FIG. 14—Modulated Differential Scanning Calorimetry (MDSC) Curve of FormB RTA 402. The section of the curve shown in the expanded view isconsistent with a glass transition temperature (T_(g)).

FIG. 15—Improved Bioavailability of Form B (Amorphous) in CynomolgusMonkeys. The figure shows a representative plot of the area under thecurve for Form A and Form B, following a 4.1 mg/kg oral administrationto cynomolgus monkeys. Each data point represents the mean plasmaconcentration of CDDO methyl ester in 8 animals. Error bars representthe standard deviation within the sampled population.

DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS I. The Present Invention

The present invention concerns new methods for the treatment andprevention of renal disease and related disorders, including diabetesand cardiovascular disease, involving the use of triterpenoids.

II. Definitions

As used herein, the term “amino” means —NH₂; the term “nitro” means—NO₂; the term “halo” designates —F, —Cl, —Br or —I; the term “mercapto”means —SH; the term “cyano” means —CN; the term “silyl” means —SiH₃, andthe term “hydroxy” means —OH.

The term “heteroatom-substituted,” when used to modify a class oforganic radicals (e.g., alkyl, aryl, acyl, etc.), means that one, ormore than one, hydrogen atom of that radical has been replaced by aheteroatom, or a heteroatom containing group. Examples of heteroatomsand heteroatom containing groups include: hydroxy, cyano, alkoxy, ═O,═S, —NO₂, —N(CH₃)₂, amino, or —SH. Specific heteroatom-substitutedorganic radicals are defined more fully below.

The term “heteroatom-unsubstituted,” when used to modify a class oforganic radicals (e.g., alkyl, aryl, acyl, etc.) means that none of thehydrogen atoms of that radical have been replaced with a heteroatom or aheteroatom containing group. Substitution of a hydrogen atom with acarbon atom, or a group consisting of only carbon and hydrogen atoms, isnot sufficient to make a group heteroatom-substituted. For example, thegroup —C₆H₄C≡CH is an example of a heteroatom-unsubstituted aryl group,while —C₆H₄F is an example of a heteroatom-substituted aryl group.Specific heteroatom-unsubstituted organic radicals are defined morefully below.

The term “alkyl” includes straight-chain alkyl groups, branched-chainalkyl groups, cycloalkyl (alicyclic) groups, alkylheteroatom-substituted cycloalkyl groups, and cycloalkylheteroatom-substituted alkyl groups. The term “heteroatom-unsubstitutedC_(n)-alkyl” refers to a radical having a linear or branched, cyclic oracyclic structure, further having no carbon-carbon double or triplebonds, further having a total of n carbon atoms, all of which arenonaromatic, 3 or more hydrogen atoms, and no heteroatoms. For example,a heteroatom-unsubstituted C₁-C₁₀-alkyl has 1 to 10 carbon atoms. Thegroups, —CH₃, —CH₂CH₃, —CH₂CH₂CH₃, —CH(CH₃)₂, —CH(CH₂)₂ (cyclopropyl),—CH₂CH₂CH₂CH₃, —CH(CH₃)CH₂CH₃, —CH₂CH(CH₃)₂, —C(CH₃)₃, —CH₂C(CH₃)₃,cyclobutyl, cyclopentyl, and cyclohexyl, are all examples ofheteroatom-unsubstituted alkyl groups. The term “heteroatom-substitutedC_(n)-alkyl” refers to a radical having a single saturated carbon atomas the point of attachment, no carbon-carbon double or triple bonds,further having a linear or branched, cyclic or acyclic structure,further having a total of n carbon atoms, all of which are nonaromatic,0, 1, or more than one hydrogen atom, at least one heteroatom, whereineach heteroatom is independently selected from the group consisting ofN, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substitutedC₁-C₁₀-alkyl has 1 to 10 carbon atoms. The following groups are allexamples of heteroatom-substituted alkyl groups: trifluoromethyl, —CH₂F,—CH₂Cl, —CH₂Br, —CH₂OH, —CH₂OCH₃, —CH₂OCH₂CH₃, —CH₂OCH₂CH₂CH₃,—CH₂OCH(CH₃)₂, —CH₂OCH(CH₂)₂, —CH₂OCH₂CF₃, —CH₂OCOCH₃, —CH₂NH₂,—CH₂NHCH₃, —CH₂N(CH₃)₂, —CH₂NHCH₂CH₃, —CH₂N(CH₃)CH₂CH₃, —CH₂NHCH₂CH₂CH₃,—CH₂NHCH(CH₃)₂, —CH₂NHCH(CH₂)₂, —CH₂N(CH₂CH₃)₂, —CH₂CH₂F, —CH₂CH₂Cl,—CH₂CH₂Br, —CH₂CH₂I, —CH₂CH₂OH, —CH₂CH₂OCOCH₃, —CH₂CH₂NH₂,—CH₂CH₂N(CH₃)₂, —CH₂CH₂NHCH₂CH₃, —CH₂CH₂N(CH₃)CH₂CH₃,—CH₂CH₂NHCH₂CH₂CH₃, —CH₂CH₂NHCH(CH₃)₂, —CH₂CH₂NHCH(CH₂)₂,—CH₂CH₂N(CH₂CH₃)₂, —CH₂CH₂NHCO₂C(CH₃)₃, and —CH₂Si(CH₃)₃.

The term “heteroatom-unsubstituted C_(n)-alkenyl” refers to a radicalhaving a linear or branched, cyclic or acyclic structure, further havingat least one nonaromatic carbon-carbon double bond, but no carbon-carbontriple bonds, a total of n carbon atoms, three or more hydrogen atoms,and no heteroatoms. For example, a heteroatom-unsubstitutedC₂-C₁₀-alkenyl has 2 to 10 carbon atoms. Heteroatom-unsubstitutedalkenyl groups include: —CH═CH₂, —CH═CHCH₃, —CH═CHCH₂CH₃,—CH═CHCH₂CH₂CH₃, —CH═CHCH(CH₃)₂, —CH═CHCH(CH₂)₂, —CH₂CH═CH₂,—CH₂CH═CHCH₃, —CH₂CH═CHCH₂CH₃, —CH₂CH═CHCH₂CH₂CH₃, —CH₂CH═CHCH(CH₃)₂,—CH₂CH═CHCH(CH₂)₂, and —CH═CH—C₆H₅. The term “heteroatom-substitutedC_(n)-alkenyl” refers to a radical having a single nonaromatic carbonatom as the point of attachment and at least one nonaromaticcarbon-carbon double bond, but no carbon-carbon triple bonds, furtherhaving a linear or branched, cyclic or acyclic structure, further havinga total of n carbon atoms, 0, 1, or more than one hydrogen atom, and atleast one heteroatom, wherein each heteroatom is independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Forexample, a heteroatom-substituted C₂-C₁₀-alkenyl has 2 to 10 carbonatoms. The groups, —CH═CHF, —CH═CHCl and —CH═CHBr, are examples ofheteroatom-substituted alkenyl groups.

The term “heteroatom-unsubstituted C_(n)-alkynyl” refers to a radicalhaving a linear or branched, cyclic or acyclic structure, further havingat least one carbon-carbon triple bond, a total of n carbon atoms, atleast one hydrogen atom, and no heteroatoms. For example, aheteroatom-unsubstituted C₂-C₁₀-alkynyl has 2 to 10 carbon atoms. Thegroups, —C≡CH, —C≡CCH₃, and —C≡CC₆H₅ are examples ofheteroatom-unsubstituted alkynyl groups. The term“heteroatom-substituted C_(n)-alkynyl” refers to a radical having asingle nonaromatic carbon atom as the point of attachment and at leastone carbon-carbon triple bond, further having a linear or branched,cyclic or acyclic structure, and having a total of n carbon atoms, 0, 1,or more than one hydrogen atom, and at least one heteroatom, whereineach heteroatom is independently selected from the group consisting ofN, O, F, Cl, Br, I, Si, P, and S. For example, a heteroatom-substitutedC₂-C₁₀-alkynyl has 2 to 10 carbon atoms. The group, —C≡CSi(CH₃)₃, is anexample of a heteroatom-substituted alkynyl group.

The term “heteroatom-unsubstituted C_(n)-aryl” refers to a radicalhaving a single carbon atom as a point of attachment, wherein the carbonatom is part of an aromatic ring structure containing only carbon atoms,further having a total of n carbon atoms, 5 or more hydrogen atoms, andno heteroatoms. For example, a heteroatom-unsubstituted C₆-C₁₀-aryl has6 to 10 carbon atoms. Examples of heteroatom-unsubstituted aryl groupsinclude phenyl, methylphenyl, (dimethyl)phenyl, —C₆H₄CH₂CH₃,—C₆H₄CH₂CH₂CH₃, —C₆H₄CH(CH₃)₂, —C₆H₄CH(CH₂)₂, —C₆H₃ (CH₃)CH₂CH₃,—C₆H₄CH═CH₂, —C₆H₄CH═CHCH₃, —C₆H₄C≡CH, —C₆H₄C≡CCH₃, naphthyl, and theradical derived from biphenyl. The term “heteroatom-unsubstituted aryl”includes carbocyclic aryl groups, biaryl groups, and radicals derivedfrom polycyclic fused hydrocarbons (PAHs). The term“heteroatom-substituted C_(n)-aryl” refers to a radical having either asingle aromatic carbon atom or a single aromatic heteroatom as the pointof attachment, further having a total of n carbon atoms, at least onehydrogen atom, and at least one heteroatom, further wherein eachheteroatom is independently selected from the group consisting of N, O,F, Cl, Br, I, Si, P, and S. For example, a heteroatom-unsubstitutedC₁-C₁₀-heteroaryl has 1 to 10 carbon atoms. The term“heteroatom-substituted aryl” includes heteroaryl groups. It alsoincludes those groups derived from the compounds: pyrrole, furan,thiophene, imidazole, oxazole, isoxazole, thiazole, isothiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine, pyrimidine, and thelike. Further examples of heteroatom-substituted aryl groups include thegroups: —C₆H₄F, —C₆H₄Cl, —C₆H₄Br, —C₆H₄₁, —C₆H₄OH, —C₆H₄OCH₃,—C₆H₄OCH₂CH₃, —C₆H₄OCOCH₃, —C₆H₄OC₆H₅, —C₆H₄NH₂, —C₆H₄NHCH₃,—C₆H₄NHCH₂CH₃, —C₆H₄CH₂Cl, —C₆H₄CH₂Br, —C₆H₄CH₂OH, —C₆H₄CH₂OCOCH₃,—C₆H₄CH₂NH₂, —C₆H₄N(CH₃)₂, —C₆H₄CH₂CH₂Cl, —C₆H₄CH₂CH₂OH,—C₆H₄CH₂CH₂OCOCH₃, —C₆H₄CH₂CH₂NH₂, —C₆H₄CH₂CH═CH₂, —C₆H₄CF₃, —C₆H₄CN,—C₆H₄C≡CSi(CH₃)₃, —C₆H₄COH, —C₆H₄COCH₃, —C₆H₄COCH₂CH₃, —C₆H₄COCH₂CF₃,—C₆H₄COC₆H₅, —C₆H₄CO₂H, —C₆H₄CO₂CH₃, —C₆H₄CONH₂, —C₆H₄CONHCH₃,—C₆H₄CON(CH₃)₂, furanyl, thienyl, pyridyl, pyrrolyl, pyrimidyl,pyrazinyl, imidazoyl, quinolyl and indolyl.

The term “heteroatom-unsubstituted C_(n)-aralkyl” refers to a radicalhaving a single saturated carbon atom as the point of attachment,further having a total of n carbon atoms, wherein at least 6 of thecarbon atoms form an aromatic ring structure containing only carbonatoms, 7 or more hydrogen atoms, and no heteroatoms. For example, aheteroatom-unsubstituted C₇-C₁₀-aralkyl has 7 to 10 carbon atoms.Examples of heteroatom-unsubstituted aralkyls include phenylmethyl(benzyl) and phenylethyl. The term “heteroatom-substitutedC_(n)-aralkyl” refers to a radical having a single saturated carbon atomas the point of attachment, further having a total of n carbon atoms, 0,1, or more than one hydrogen atom, and at least one heteroatom, whereinat least one of the carbon atoms is incorporated in an aromatic ringstructure, further wherein each heteroatom is independently selectedfrom the group consisting of N, O, F, Cl, Br, I, Si, P, and S. Forexample, a heteroatom-substituted C₂-C₁₀-heteroaralkyl has 2 to 10carbon atoms.

The term “heteroatom-unsubstituted C_(n)-acyl” refers to a radicalhaving a single carbon atom of a carbonyl group as the point ofattachment, further having a linear or branched, cyclic or acyclicstructure, further having a total of n carbon atoms, 1 or more hydrogenatoms, a total of one oxygen atom, and no additional heteroatoms. Forexample, a heteroatom-unsubstituted C₁-C₁₀-acyl has 1 to 10 carbonatoms. The groups, —COH, —COCH₃, —COCH₂CH₃, —COCH₂CH₂CH₃, —COCH(CH₃)₂,—COCH(CH₂)₂, —COC₆H₅, —COC₆H₄CH₃, —COC₆H₄CH₂CH₃, —COC₆H₄CH₂CH₂CH₃,—COC₆H₄CH(CH₃)₂, —COC₆H₄CH(CH₂)₂, and —COC₆H₃(CH₃)₂, are examples ofheteroatom-unsubstituted acyl groups. The term “heteroatom-substitutedC_(n)-acyl” refers to a radical having a single carbon atom as the pointof attachment, the carbon atom being part of a carbonyl group, furtherhaving a linear or branched, cyclic or acyclic structure, further havinga total of n carbon atoms, 0, 1, or more than one hydrogen atom, atleast one additional heteroatom in addition to the oxygen of thecarbonyl group, wherein each additional heteroatom is independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.For example, a heteroatom-substituted C₁-C₁₀-acyl has 1 to 10 carbonatoms. The term heteroatom-substituted acyl includes carbamoyl,thiocarboxylate, and thiocarboxylic acid groups. The groups, —COCH₂CF₃,—CO₂H, —CO₂CH₃, —CO₂CH₂CH₃, —CO₂CH₂CH₂CH₃, —CO₂CH(CH₃)₂, —CO₂CH(CH₂)₂,—CONH₂, —CONHCH₃, —CONHCH₂CH₃, —CONHCH₂CH₂CH₃, —CONHCH(CH₃)₂,—CONHCH(CH₂)₂, —CON(CH₃)₂, —CON(CH₂CH₃)CH₃, —CON(CH₂CH₃)₂ and—CONHCH₂CF₃, are examples of heteroatom-substituted acyl groups.

The term “heteroatom-unsubstituted C_(n)-alkoxy” refers to a group,having the structure —OR, in which R is a heteroatom-unsubstitutedC_(n)-alkyl, as that term is defined above. Heteroatom-unsubstitutedalkoxy groups include: —OCH₃, —OCH₂CH₃, —OCH₂CH₂CH₃, —OCH(CH₃)₂, and—OCH(CH₂)₂. The term “heteroatom-substituted C_(n)-alkoxy” refers to agroup, having the structure —OR, in which R is a heteroatom-substitutedC_(n)-alkyl, as that term is defined above. For example, —OCH₂CF₃ is aheteroatom-substituted alkoxy group.

The term “heteroatom-unsubstituted C_(n)-alkenyloxy” refers to a group,having the structure —OR, in which R is a heteroatom-unsubstitutedC_(n)-alkenyl, as that term is defined above. The term“heteroatom-substituted C_(n)-alkenyloxy” refers to a group, having thestructure —OR, in which R is a heteroatom-substituted C_(n)-alkenyl, asthat term is defined above.

The term “heteroatom-unsubstituted C_(n)-alkynyloxy” refers to a group,having the structure —OR, in which R is a heteroatom-unsubstitutedC_(n)-alkynyl, as that term is defined above. The term“heteroatom-substituted C_(n)-alkynyloxy” refers to a group, having thestructure —OR, in which R is a heteroatom-substituted C_(n)-alkynyl, asthat term is defined above.

The term “heteroatom-unsubstituted C_(n)-aryloxy” refers to a group,having the structure —OAr, in which Ar is a heteroatom-unsubstitutedC_(n)-aryl, as that term is defined above. An example of aheteroatom-unsubstituted aryloxy group is —OC₆H₅. The term“heteroatom-substituted C_(n)-aryloxy” refers to a group, having thestructure —OAr, in which Ar is a heteroatom-substituted C_(n)-aryl, asthat term is defined above.

The term “heteroatom-unsubstituted C_(n)-aralkyloxy” refers to a group,having the structure —OR_(Ar), in which R_(Ar) is aheteroatom-unsubstituted C_(n)-aralkyl, as that term is defined above.The term “heteroatom-substituted C_(n)-aralkyloxy” refers to a group,having the structure —OR_(Ar), in which R_(Ar) is aheteroatom-substituted C_(n)-aralkyl, as that term is defined above.

The term “heteroatom-unsubstituted C_(n)-acyloxy” refers to a group,having the structure —OAc, in which Ac is a heteroatom-unsubstitutedC_(n)-acyl, as that term is defined above. A heteroatom-unsubstitutedacyloxy group includes alkylcarbonyloxy and arylcarbonyloxy groups. Forexample, —OCOCH₃ is an example of a heteroatom-unsubstituted acyloxygroup. The term “heteroatom-substituted C_(n)-acyloxy” refers to agroup, having the structure —OAc, in which Ac is aheteroatom-substituted C_(n)-acyl, as that term is defined above. Aheteroatom-substituted acyloxy group includes alkoxycarbonyloxy,aryloxycarbonyloxy, carboxylate, alkylcarbonyl, alkoxycarbonyl,aminocarbonyl, and alkylthiocarbonyl groups.

The term “heteroatom-unsubstituted C_(n)-alkylamino” refers to a radicalhaving a single nitrogen atom as the point of attachment, further havingone or two saturated carbon atoms attached to the nitrogen atom, furtherhaving a linear or branched, cyclic or acyclic structure, containing atotal of n carbon atoms, all of which are nonaromatic, 4 or morehydrogen atoms, a total of 1 nitrogen atom, and no additionalheteroatoms. For example, a heteroatom-unsubstituted C₁-C₁₀-alkylaminohas 1 to 10 carbon atoms. The term “heteroatom-unsubstitutedC_(n)-alkylamino” includes groups, having the structure —NHR, in which Ris a heteroatom-unsubstituted C_(n)-alkyl, as that term is definedabove. A heteroatom-unsubstituted alkylamino group would include —NHCH₃,—NHCH₂CH₃, —NHCH₂CH₂CH₃, —NHCH(CH₃)₂, —NHCH(CH₂)₂, —NHCH₂CH₂CH₂CH₃,—NHCH(CH₃)CH₂CH₃, —NHCH₂CH(CH₃)₂, —NHC(CH₃)₃, —N(CH₃)₂, —N(CH₃)CH₂CH₃,—N(CH₂CH₃)₂, N-pyrrolidinyl, and N-piperidinyl. The term“heteroatom-substituted C_(n)-alkylamino” refers to a radical having asingle nitrogen atom as the point of attachment, further having one ortwo saturated carbon atoms attached to the nitrogen atom, nocarbon-carbon double or triple bonds, further having a linear orbranched, cyclic or acyclic structure, further having a total of ncarbon atoms, all of which are nonaromatic, 0, 1, or more than onehydrogen atom, and at least one additional heteroatom, that is, inaddition to the nitrogen atom at the point of attachment, wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₁-C₁₀-alkylamino has 1 to 10 carbon atoms. Theterm “heteroatom-substituted C_(n)-alkylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-substituted C_(n)-alkyl,as that term is defined above.

The term “heteroatom-unsubstituted C_(n)-alkenylamino” refers to aradical having a single nitrogen atom as the point of attachment,further having one or two carbon atoms attached to the nitrogen atom,further having a linear or branched, cyclic or acyclic structure,containing at least one nonaromatic carbon-carbon double bond, a totalof n carbon atoms, 4 or more hydrogen atoms, a total of one nitrogenatom, and no additional heteroatoms. For example, aheteroatom-unsubstituted C₂-C₁₀-alkenylamino has 2 to 10 carbon atoms.The term “heteroatom-unsubstituted C_(n)-alkenylamino” includes groups,having the structure —NHR, in which R is a heteroatom-unsubstitutedC_(n)-alkenyl, as that term is defined above. Examples ofheteroatom-unsubstituted C-alkenylamino groups also includedialkenylamino and alkyl(alkenyl)amino groups. The term“heteroatom-substituted C_(n)-alkenylamino” refers to a radical having asingle nitrogen atom as the point of attachment and at least onenonaromatic carbon-carbon double bond, but no carbon-carbon triplebonds, further having one or two carbon atoms attached to the nitrogenatom, further having a linear or branched, cyclic or acyclic structure,further having a total of n carbon atoms, 0, 1, or more than onehydrogen atom, and at least one additional heteroatom, that is, inaddition to the nitrogen atom at the point of attachment, wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₂-C₁₀-alkenylamino has 2 to 10 carbon atoms. Theterm “heteroatom-substituted C_(n)-alkenylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-substitutedC_(n)-alkenyl, as that term is defined above.

The term “heteroatom-unsubstituted C_(n)-alkynylamino” refers to aradical having a single nitrogen atom as the point of attachment,further having one or two carbon atoms attached to the nitrogen atom,further having a linear or branched, cyclic or acyclic structure,containing at least one carbon-carbon triple bond, a total of n carbonatoms, at least one hydrogen atoms, a total of one nitrogen atom, and noadditional heteroatoms. For example, a heteroatom-unsubstitutedC₂-C₁₀-alkynylamino has 2 to 10 carbon atoms. The term“heteroatom-unsubstituted C_(n)-alkynylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-unsubstitutedC_(n)-alkynyl, as that term is defined above. An alkynylamino groupincludes dialkynylamino and alkyl(alkynyl)amino groups. The term“heteroatom-substituted C_(n)-alkynylamino” refers to a radical having asingle nitrogen atom as the point of attachment, further having one ortwo carbon atoms attached to the nitrogen atom, further having at leastone nonaromatic carbon-carbon triple bond, further having a linear orbranched, cyclic or acyclic structure, and further having a total of ncarbon atoms, 0, 1, or more than one hydrogen atom, and at least oneadditional heteroatom, that is, in addition to the nitrogen atom at thepoint of attachment, wherein each additional heteroatom is independentlyselected from the group consisting of N, O, F, Cl, Br, I, Si, P, and S.For example, a heteroatom-substituted C₂-C₁₀-alkynylamino has 2 to 10carbon atoms. The term “heteroatom-substituted C_(n)-alkynylamino”includes groups, having the structure —NHR, in which R is aheteroatom-substituted C_(n)-alkynyl, as that term is defined above.

The term “heteroatom-unsubstituted C_(n)-arylamino” refers to a radicalhaving a single nitrogen atom as the point of attachment, further havingat least one aromatic ring structure attached to the nitrogen atom,wherein the aromatic ring structure contains only carbon atoms, furtherhaving a total of n carbon atoms, 6 or more hydrogen atoms, a total ofone nitrogen atom, and no additional heteroatoms. For example, aheteroatom-unsubstituted C₆-C₁₀-arylamino has 6 to 10 carbon atoms. Theterm “heteroatom-unsubstituted C_(n)-arylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-unsubstituted C_(n)-aryl,as that term is defined above. A heteroatom-unsubstituted arylaminogroup includes diarylamino and alkyl(aryl)amino groups. The term“heteroatom-substituted C_(n)-arylamino” refers to a radical having asingle nitrogen atom as the point of attachment, further having a totalof n carbon atoms, at least one hydrogen atom, at least one additionalheteroatoms, that is, in addition to the nitrogen atom at the point ofattachment, wherein at least one of the carbon atoms is incorporatedinto one or more aromatic ring structures, further wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₆-C₁₀-arylamino has 6 to 10 carbon atoms. Theterm “heteroatom-substituted C_(n)-arylamino” includes groups, havingthe structure —NHR, in which R is a heteroatom-substituted C_(n)-aryl,as that term is defined above. A heteroatom-substituted arylamino groupincludes heteroarylamino groups.

The term “heteroatom-unsubstituted C_(n)-aralkylamino” refers to aradical having a single nitrogen atom as the point of attachment,further having one or two saturated carbon atoms attached to thenitrogen atom, further having a total of n carbon atoms, wherein atleast 6 of the carbon atoms form an aromatic ring structure containingonly carbon atoms, 8 or more hydrogen atoms, a total of one nitrogenatom, and no additional heteroatoms. For example, aheteroatom-unsubstituted C₇-C₁₀-aralkylamino has 7 to 10 carbon atoms.The term “heteroatom-unsubstituted C_(n)-aralkylamino” includes groups,having the structure —NHR, in which R is a heteroatom-unsubstitutedC_(n)-aralkyl, as that term is defined above. An aralkylamino groupincludes diaralkylamino groups. The term “heteroatom-substitutedC_(n)-aralkylamino” refers to a radical having a single nitrogen atom asthe point of attachment, further having at least one or two saturatedcarbon atoms attached to the nitrogen atom, further having a total of ncarbon atoms, 0, 1, or more than one hydrogen atom, at least oneadditional heteroatom, that is, in addition to the nitrogen atom at thepoint of attachment, wherein at least one of the carbon atomincorporated into an aromatic ring, further wherein each heteroatom isindependently selected from the group consisting of N, O, F, Cl, Br, I,Si, P, and S. For example, a heteroatom-substituted C₇-C₁₀-aralkylaminohas 7 to 10 carbon atoms. The term “heteroatom-substitutedC_(n)-aralkylamino” includes groups, having the structure —NHR, in whichR is a heteroatom-substituted C_(n)-aralkyl, as that term is definedabove. The term “heteroatom-substituted aralkylamino” includes the term“heteroaralkylamino.”

The term amido includes N-alkyl-amido, N-aryl-amido, N-aralkyl-amido,acylamino, alkylcarbonylamino, arylcarbonylamino, and ureido groups. Thegroup, —NHCOCH₃, is an example of a heteroatom-unsubstituted amidogroup. The term “heteroatom-unsubstituted C_(n)-amido” refers to aradical having a single nitrogen atom as the point of attachment,further having a carbonyl group attached via its carbon atom to thenitrogen atom, further having a linear or branched, cyclic or acyclicstructure, further having a total of n carbon atoms, 1 or more hydrogenatoms, a total of one oxygen atom, a total of one nitrogen atom, and noadditional heteroatoms. For example, a heteroatom-unsubstitutedC₁-C₁₀-amido has 1 to 10 carbon atoms. The term“heteroatom-unsubstituted C_(n)-amido” includes groups, having thestructure —NHR, in which R is a heteroatom-unsubstituted C_(n)-acyl, asthat term is defined above. The term “heteroatom-substitutedC_(n)-amido” refers to a radical having a single nitrogen atom as thepoint of attachment, further having a carbonyl group attached via itscarbon atom to the nitrogen atom, further having a linear or branched,cyclic or acyclic structure, further having a total of n aromatic ornonaromatic carbon atoms, 0, 1, or more than one hydrogen atom, at leastone additional heteroatom in addition to the oxygen of the carbonylgroup and the nitrogen atom at the point of attachment, wherein eachadditional heteroatom is independently selected from the groupconsisting of N, O, F, Cl, Br, I, Si, P, and S. For example, aheteroatom-substituted C₁-C₁₀-amido has 1 to 10 carbon atoms. The term“heteroatom-substituted C_(n)-amido” includes groups, having thestructure —NHR, in which R is a heteroatom-unsubstituted C_(n)-acyl, asthat term is defined above. The group, —NHCO₂CH₃, is an example of aheteroatom-substituted amido group.

In addition, atoms making up the compounds of the present invention areintended to include all isotopic forms of such atoms. Isotopes, as usedherein, include those atoms having the same atomic number but differentmass numbers. By way of general example and without limitation, isotopesof hydrogen include tritium and deuterium, and isotopes of carboninclude ¹³C and ¹⁴C. Similarly, it is contemplated that one or morecarbon atom(s) of a compound of the present invention may be replaced bya silicon atom(s). Similarly, it is contemplated that one or more oxygenatom(s) of a compound of the present invention may be replaced by asulfur or a selenium atom(s).

Any undefined valency on an atom of a structure shown in thisapplication implicitly represents a hydrogen atom bonded to the atom.

The use of the word “a” or “an,” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

Throughout this application, the term “about” is used to indicate that avalue includes the inherent variation of error for the device, themethod being employed to determine the value, or the variation thatexists among the study subjects.

The terms “comprise,” “have” and “include” are open-ended linking verbs.Any forms or tenses of one or more of these verbs, such as “comprises,”“comprising,” “has,” “having,” “includes” and “including,” are alsoopen-ended. For example, any method that “comprises,” “has” or“includes” one or more steps is not limited to possessing only those oneor more steps and also covers other unlisted steps.

The term “effective,” as that term is used in the specification and/orclaims, means adequate to accomplish a desired, expected, or intendedresult.

The term “hydrate” when used as a modifier to a compound means that thecompound has less than one (e.g., hemihydrate), one (e.g., monohydrate),or more than one (e.g., dihydrate) water molecules associated with eachcompound molecule, such as in solid forms of the compound.

As used herein, the term “IC₅₀” refers to an inhibitory dose which is50% of the maximum response obtained.

An “isomer” of a first compound is a separate compound in which eachmolecule contains the same constituent atoms as the first compound, butwhere the configuration of those atoms in three dimensions differs.

As used herein, the term “patient” or “subject” refers to a livingmammalian organism, such as a human, monkey, cow, sheep, goat, dog, cat,mouse, rat, guinea pig, or transgenic species thereof. In certainembodiments, the patient or subject is a primate. Non-limiting examplesof human subjects are adults, juveniles, infants and fetuses.

“Pharmaceutically acceptable” means that which is useful in preparing apharmaceutical composition that is generally safe, non-toxic and neitherbiologically nor otherwise undesirable and includes that which isacceptable for veterinary use as well as human pharmaceutical use.

“Pharmaceutically acceptable salts” means salts of compounds of thepresent invention which are pharmaceutically acceptable, as definedabove, and which possess the desired pharmacological activity. Suchsalts include acid addition salts formed with inorganic acids such ashydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid,phosphoric acid, and the like; or with organic acids such as1,2-ethanedisulfonic acid, 2-hydroxyethanesulfonic acid,2-naphthalenesulfonic acid, 3-phenylpropionic acid,4,4′-methylenebis(3-hydroxy-2-ene-1-carboxylic acid),4-methylbicyclo[2.2.2]oct-2-ene-1-carboxylic acid, acetic acid,aliphatic mono- and dicarboxylicacids, aliphatic sulfuric acids,aromatic sulfuric acids, benzenesulfonic acid, benzoic acid,camphorsulfonic acid, carbonic acid, cinnamic acid, citric acid,cyclopentanepropionic acid, ethanesulfonic acid, fumaric acid,glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid,heptanoic acid, hexanoic acid, hydroxynaphthoic acid, lactic acid,laurylsulfuric acid, maleic acid, malic acid, malonic acid, mandelicacid, methanesulfonic acid, muconic acid, o-(4-hydroxybenzoyl)benzoicacid, oxalic acid, p-chlorobenzenesulfonic acid, phenyl-substitutedalkanoic acids, propionic acid, p-toluenesulfonic acid, pyruvic acid,salicylic acid, stearic acid, succinic acid, tartaric acid,tertiarybutylacetic acid, trimethylacetic acid, and the like.Pharmaceutically acceptable salts also include base addition salts whichmay be formed when acidic protons present are capable of reacting withinorganic or organic bases. Acceptable inorganic bases include sodiumhydroxide, sodium carbonate, potassium hydroxide, aluminum hydroxide andcalcium hydroxide. Acceptable organic bases include ethanolamine,diethanolamine, triethanolamine, tromethamine, N-methylglucamine and thelike. It should be recognized that the particular anion or cationforming a part of any salt of this invention is not critical, so long asthe salt, as a whole, is pharmacologically acceptable. Additionalexamples of pharmaceutically acceptable salts and their methods ofpreparation and use are presented in Handbook of Pharmaceutical SaltsProperties, and Use (P. H. Stahl & C. G. Wermuth eds., Verlag HelveticaChimica Acta, 2002),

As used herein, “predominantly one enantiomer” means that a compoundcontains at least about 85% of one enantiomer, or more preferably atleast about 90% of one enantiomer, or even more preferably at leastabout 95% of one enantiomer, or most preferably at least about 99% ofone enantiomer. Similarly, the phrase “substantially free from otheroptical isomers” means that the composition contains at most about 15%of another enantiomer or diastereomer, more preferably at most about 10%of another enantiomer or diastereomer, even more preferably at mostabout 5% of another enantiomer or diastereomer, and most preferably atmost about 1% of another enantiomer or diastereomer.

“Prevention” or “preventing” includes: (1) inhibiting the onset of adisease in a subject or patient which may be at risk and/or predisposedto the disease but does not yet experience or display any or all of thepathology or symptomatology of the disease, and/or (2) slowing the onsetof the pathology or symptomatology of a disease in a subject or patientwhich may be at risk and/or predisposed to the disease but does not yetexperience or display any or all of the pathology or symptomatology ofthe disease.

The term “saturated” when referring to an atom means that the atom isconnected to other atoms only by means of single bonds.

A “stereoisomer” or “optical isomer” is an isomer of a given compound inwhich the same atoms are bonded to the same other atoms, but where theconfiguration of those atoms in three dimensions differs. “Enantiomers”are stereoisomers of a given compound that are mirror images of eachother, like left and right hands. “Diastereomers” are stereoisomers of agiven compound that are not enantiomers.

“Therapeutically effective amount” or “pharmaceutically effectiveamount” means that amount which, when administered to a subject orpatient for treating a disease, is sufficient to effect such treatmentfor the disease.

“Treatment” or “treating” includes (1) inhibiting a disease in a subjector patient experiencing or displaying the pathology or symptomatology ofthe disease (e.g., arresting further development of the pathology and/orsymptomatology), (2) ameliorating a disease in a subject or patient thatis experiencing or displaying the pathology or symptomatology of thedisease (e.g., reversing the pathology and/or symptomatology), and/or(3) effecting any measurable decrease in a disease in a subject orpatient that is experiencing or displaying the pathology orsymptomatology of the disease.

As used herein, the term “water soluble” means that the compounddissolves in water at least to the extent of 0.010 mole/liter or isclassified as soluble according to literature precedence.

Other abbreviations used herein are as follows: DMSO, dimethylsulfoxide; NO, nitric oxide; iNOS, inducible nitric oxide synthase;COX-2, cyclooxygenase-2; NGF, nerve growth factor; IBMX,isobutylmethylxanthine; FBS, fetal bovine serum; GPDH, glycerol3-phosphate dehydrogenase; RXR, retinoid X receptor; TGF-β, transforminggrowth factor-β; IFNγ or IFN-γ, interferon-γ; LPS, bacterial endotoxiclipopolysaccharide; TNFα or TNF-α, tumor necrosis factor-α; IL-1β,interleukin-1β; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; MTBE,methyl-tert-butylether; MTT,3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide; TCA,trichloroacetic acid; HO-1, inducible heme oxygenase.

The above definitions supersede any conflicting definition in any of thereference that is incorporated by reference herein.

III. Synthetic Triterpenoids

Triterpenoids, biosynthesized in plants by the cyclization of squalene,are used for medicinal purposes in many Asian countries; and some, likeursolic and oleanolic acids, are known to be anti-inflammatory andanti-carcinogenic (Huang et al., 1994; Nishino et al., 1988). However,the biological activity of these naturally-occurring molecules isrelatively weak, and therefore the synthesis of new analogs to enhancetheir potency was undertaken (Honda et al., 1997; Honda et al., 1998).Subsequent research has identified a number of synthetic compounds thathave improved activity as compared to the naturally-occurringtriterpenoids.

The ongoing efforts for the improvement of anti-inflammatory andantiproliferative activity of oleanolic and ursolic acid analogs led tothe discovery of 2-cyano-3,12-dioxooleane-1,9(11)-dien-28-oic acid(CDDO, RTA 402) and related compounds (e.g., CDDO-Me, TP-225, CDDO-Im)(Honda et al., 1997, 1998, 1999, 2000a, 2000b, 2002; Suh et al., 1998;1999; 2003; Place et al., 2003; Liby et al., 2005). In the case ofinducing cytoprotective genes through Keap1-Nrf2-antioxidant responseelement (ARE) signaling, a recent structure activity evaluation of 15triterpenoids noted the importance of Michael acceptor groups on boththe A and C rings, a nitrile group at C-2 of the A ring, and thatsubstituents at C-17 affected pharmacodynamic action in vivo (Yates etal., 2007).

In general, CDDO is the prototype for a large number of compounds in afamily of agents that have been shown useful in a variety of contexts.For example, CDDO-Me and CDDO-Im are reported to possess the ability tomodulate transforming growth factor-β (TGF-β)/Smad signaling in severaltypes of cells (Suh et al., 2003; Minns et al., 2004; Mix et al., 2004).Both are known to be potent inducers of heme-oxygenase-1 and Nrf2/AREsignaling (Liby et al., 2005), and a series of synthetic triterpenoid(TP) analogs of oleanolic acid have also been shown to be potentinducers of the phase 2 response, that is elevation of NAD(P)H-quinoneoxidoreductase and heme oxygenase 1 (HO-1), which is a major protectorof cells against oxidative and electrophile stress (Dinkova-Kostova etal., 2005). Like previously identified phase 2 inducers, the TP analogswere shown to use the antioxidant response element-Nrf2-Keap1 signalingpathway.

RTA 402 (bardoxolone methyl), one of the compounds for use with themethods of this invention, is an Antioxidant Inflammation Modulator(AIM) in clinical development for inflammation and cancer-relatedindications that inhibits immune-mediated inflammation by restoringredox homeostasis in inflamed tissues. It induces the cytoprotectivetranscription factor Nrf2 and suppresses the activities of thepro-oxidant and pro-inflammatory transcription factors NF-κB and STAT3.In vivo, RTA 402 has demonstrated significant single agentanti-inflammatory activity in several animal models of inflammation suchas renal damage in the cisplatin model and acute renal injury in theischemia-reperfusion model. In addition, significant reductions in serumcreatinine have been observed in patients treated with RTA 402.

In one aspect of the invention, the compounds of the present inventionmay be used for treating a subject having a renal disease or conditioncaused by elevated levels of oxidative stress in one or more tissues.The oxidative stress may be accompanied by either acute or chronicinflammation. The oxidative stress may be caused by acute exposure to anexternal agent such as ionizing radiation or a cytotoxic chemotherapyagent (e.g., doxorubicin), by trauma or other acute tissue injury, byischemia/reperfusion injury, by poor circulation or anemia, by localizedor systemic hypoxia or hyperoxia, or by other abnormal physiologicalstates such as hyperglycemia or hypoglycemia.

Accordingly, in pathologies involving oxidative stress alone oroxidative stress exacerbated by inflammation, treatment may compriseadministering to a subject a therapeutically effective amount of acompound of this invention, such as those described above or throughoutthis specification. Treatment may be administered preventively inadvance of a predictable state of oxidative stress (e.g., organtransplantation or the administration of therapy to a cancer patient),or it may be administered therapeutically in settings involvingestablished oxidative stress and inflammation.

Newer amide derivatives of CDDO have now also been found to be promisingagents, for example for their ability to pass through the blood brainbarrier. In addition to the methyl amide of CDDO (CDDO-MA), as reportedin Honda et al. (2002), the invention provides for the use of additionalCDDO amide derivatives, such as the ethyl amide (CDDO-EA), as wellfluorinated amide derivatives of CDDO, such as the 2,2,2-trifluoroethylamide derivative of CDDO (CDDO-TFEA).

The compounds of the present invention can be prepared according to themethods taught by Honda et al. (1998), Honda et al. (2000b), Honda etal. (2002), Yates et al. (2007), and U.S. Pat. Nos. 6,326,507 and6,974,801, which are all incorporated herein by reference.

Non-limiting examples of triterpenoids that may be used in accordancewith the methods of this invention are shown here.

The compounds for use with the present invention, such as those of thetable above, are structurally similar to RTA 402 and in many casesexhibit similar biological properties, as has been noted above. Asadditional examples, Table 1 summarizes in vitro results for several ofthese compounds in which RAW264.7 macrophages were pre-treated with DMSOor drugs at various concentrations (nM) for 2 hours, then treated with20 ng/ml IFNγ for 24 hours. NO concentration in media was determinedusing a Griess reagent system; cell viability was determined using WST-1reagent. NQO1 CD represents the concentration required to induce atwo-fold increase in the expression of NQO1, an Nrf2-regulatedantioxidant enzyme, in Hepa1c1c7 murine hepatoma cells (Dinkova-Kostovaet al., 2005). All these results are orders of magnitude more activethan, for example, the parent oleanolic acid molecule. In part becauseinduction of antioxidant pathways resulting from Nrf2 activationprovides important protective effects against oxidative stress andinflammation, compounds related to RTA 402 may also provide significantbenefits similar to those presented for RTA 402 in this application, andthese related compounds may, therefore, be used for the treatment and/orprevention of diseases, such as: renal/kidney disease (RKD), insulinresistance, diabetes, endothelial dysfunction, fatty liver disease,cardiovascular disease (CVD), and related disorders.

TABLE 1 Suppression of IFNγ-induced NO production. RAW264.7 Hepa1c1c7(20 ng/ml IFNγ) cells Working ID NO IC₅₀ WST-1 IC₅₀ NQO1 CD RTA 401 ~10nM >200 nM 2.3 nM RTA 402 2.2 nM 80 nM 1.0 nM RTA 403 ~0.6 nM 100 nM 3.3nM RTA 404 5.8 nM 100 nM n/a RTA 405 6 nM ~200 nM n/a TP-225 ~0.4 nM 75nM 0.28 nM 

The synthesis of CDDO-MA is discussed in Honda et al. (2002), which isincorporated herein by reference. The syntheses of CDDO-EA and CDDO-TFEAare presented in Yates et al. (2007), which is incorporated herein byreference, and shown in the Scheme 1 below.

IV. Polymorphic Forms of CDDO-Me

Polymorphic forms of the compounds of the present invention, e.g., FormsA and B of CDDO-Me, may be used in accordance with the methods of thisinventions. Form B displays a bioavailability that is surprisinglybetter than that of Form A (FIG. 15). Specifically the bioavailabilityof Form B was higher than that of Form A CDDO-Me in monkeys when themonkeys received equivalent dosages of the two forms orally, in gelatincapsules (U.S. application Ser. No. 12/191,176, filed Aug. 13, 2008).

“Form A” of CDDO-Me (RTA 402) is unsolvated (non-hydrous) and can becharacterized by a distinctive crystal structure, with a space group ofP4₃2₁2 (no. 96) unit cell dimensions of a=14.2 Å, b=14.2 Å and c=81.6 Å,and by a packing structure, whereby three molecules are packed inhelical fashion down the crystallographic b axis. In some embodiments,Form A can also be characterized by X-ray powder diffraction (XRPD)pattern (CuKα) comprising significant diffraction peaks at about 8.8,12.9, 13.4, 14.2 and 17.4° 2θ. In some variations, the X-ray powderdiffraction of Form A is substantially as shown in FIG. 12A or FIG. 12B.

Unlike Form A, “Form B” of CDDO-Me is in a single phase but lacks such adefined crystal structure. Samples of Form B show no long-rangemolecular correlation, i.e., above roughly 20 Å. Moreover, thermalanalysis of Form B samples reveals a glass transition temperature(T_(g)) in a range from about 120° C. to about 130° C. (FIG. 14). Incontrast, a disordered nanocrystalline material does not display a T_(g)but instead only a melting temperature (T_(m)), above which crystallinestructure becomes a liquid. Form B is typified by an XRPD spectrum (FIG.12C) differing from that of Form A (FIG. 12A or FIG. 12B). Since it doesnot have a defined crystal structure, Form B likewise lacks distinctXRPD peaks, such as those that typify Form A, and instead ischaracterized by a general “halo” XRPD pattern. In particular, thenon-crystalline Form B falls into the category of “X-ray amorphous”solids because its XRPD pattern exhibits three or fewer primarydiffraction halos. Within this category, Form B is a “glassy” material.

Form A and Form B of CDDO-Me are readily prepared from a variety ofsolutions of the compound. For example, Form B can be prepared by fastevaporation or slow evaporation in MTBE, THF, toluene, or ethyl acetate.Form A can be prepared in several ways, including via fast evaporation,slow evaporation, or slow cooling of a CDDO-Me solution in ethanol ormethanol. Preparations of CDDO-Me in acetone can produce either Form A,using fast evaporation, or Form B, using slow evaporation.

Various means of characterization can be used together to distinguishForm A and Form B CDDO-Me from each other and from other forms ofCDDO-Me. Illustrative of the techniques suitable for this purpose aresolid state Nuclear Magnetic Resonance (NMR), X-ray powder diffraction(compare FIGS. 12A & B with FIG. 12C), X-ray crystallography,Differential Scanning Calorimetry (DSC) (compare FIG. 13 with FIG. 14),dynamic vapor sorption/desorption (DVS), Karl Fischer analysis (KF), hotstage microscopy, modulated differential screening calorimetry, FT-IR,and Raman spectroscopy. In particular, analysis of the XRPD and DSC datacan distinguish Form A, Form B, and a hemibenzenate forms of CDDO-Me(U.S. application Ser. No. 12/191,176, filed Aug. 13, 2008.)

Additional details regarding polymorphic forms of CDDO-Me are describedin U.S. Provisional Application No. 60/955,939, filed Aug. 15, 2007, andthe corresponding non-provisional U.S. application Ser. No. 12/191,176,filed Aug. 13, 2008, which are both incorporated herein by reference intheir entireties.

V. Use of Triterpenoids for the Treatment of Chronic Kidney Disease,Insulin Resistance/Diabetes and Endothelial Dysfunction/CardiovascularDisease

The compounds and methods of this invention may be used for treatingvarious aspects of renal/kidney disease, including both acute andchronic indications. In general, the method will comprise administeringto the subjects pharmaceutically effective amounts of a compound of thisinvention.

Inflammation contributes significantly to the pathology of chronickidney disease (CKD). There is also a strong mechanistic link betweenoxidative stress and renal dysfunction. The NF-κB signaling pathwayplays an important role in the progression of CKD as NF-κB regulates thetranscription of MCP-1, a chemokine that is responsible for therecruitment of monocytes/macrophages resulting in an inflammatoryresponse that ultimately injures the kidney (Wardle, 2001). TheKeap1/Nrf2/ARE pathway controls the transcription of several genesencoding antioxidant enzymes, including heme oxygenase-1 (HO-1).Ablation of the Nrf2 gene in female mice results in the development oflupus-like glomerular nephritis (Yoh et al., 2001; Ma et al., 2006).Furthermore, several studies have demonstrated that HO-1 expression isinduced in response to renal damage and inflammation and that thisenzyme and its products—bilirubin and carbon monoxide—play a protectiverole in the kidney (Nath et al., 2006).

The glomerulus and the surrounding Bowman's capsule constitute the basicfunctional unit of the kidney. Glomerular filtration rate (GFR) is thestandard measure of renal function. Creatinine clearance is commonlyused to measure GFR. However, the level of serum creatinine is commonlyused as a surrogate measure of creatinine clearance. For instance,excessive levels of serum creatinine are generally accepted to indicateinadequate renal function and reductions in serum creatinine over timeare accepted as an indication of improved renal function. Normal levelsof creatinine in the blood are approximately 0.6 to 1.2 milligrams (mg)per deciliter (dl) in adult males and 0.5 to 1.1 milligrams perdeciliter in adult females.

Acute kidney injury (AKI) can occur following ischemia-reperfusion,treatment with certain pharmacological agents such as cisplatin andrapamycin, and intravenous injection of radiocontrast media used inmedical imaging. As in CKD, inflammation and oxidative stress contributeto the pathology of AKI. The molecular mechanisms underlyingradiocontrast-induced nephropathy (RCN) are not well understood;however, it is likely that a combination of events including prolongedvasoconstriction, impaired kidney autoregulation, and direct toxicity ofthe contrast media all contribute to renal failure (Tumlin et al.,2006). Vasoconstriction results in decreased renal blood flow and causesischemia-reperfusion and the production of reactive oxygen species. HO-1is strongly induced under these conditions and has been demonstrated toprevent ischemia-reperfusion injury in several different organs,including the kidney (Nath et al., 2006). Specifically, induction ofHO-1 has been shown to be protective in a rat model of RCN (Goodman etal., 2007). Reperfusion also induces an inflammatory response, in partthough activation of NF-κB signaling (Nichols, 2004). Targeting NF-κBhas been proposed as a therapeutic strategy to prevent organ damage(Zingarelli et al., 2003).

Without being bound by theory, the potency of the compounds of thepresent invention, e.g., RTA 402, is largely derived from the additionof α,β-unsaturated carbonyl groups. In in vitro assays, most activity ofthe compounds can be abrogated by the introduction of dithiothreitol(DTT), N-acetyl cysteine (NAC), or glutathione (GSH); thiol containingmoieties that interact with α,β-unsaturated carbonyl groups (Wang etal., 2000; Ikeda et al., 2003; 2004; Shishodia et al., 2006).Biochemical assays have established that RTA 402 directly interacts witha critical cysteine residue (C179) on IKKβ (see below) and inhibits itsactivity (Shishodia et al., 2006; Ahmad et al., 2006). IKKβ controlsactivation of NF-κB through the “classical” pathway which involvesphosphorylation-induced degradation of IκB resulting in release of NF-κBdimers to the nucleus. In macrophages, this pathway is responsible forthe production of many pro-inflammatory molecules in response to TNFαand other pro-inflammatory stimuli.

RTA 402 also inhibits the JAK/STAT signaling pathway at multiple levels.JAK proteins are recruited to transmembrane receptors (e.g., IL-6R) uponactivation by ligands such as interferons and interleukins. JAKs thenphosphorylate the intracellular portion of the receptor causingrecruitment of STAT transcription factors. The STATs are thenphosphorylated by JAKs, form dimers, and translocate to the nucleuswhere they activate transcription of several genes involved ininflammation. RTA 402 inhibits constitutive and IL-6-induced STAT3phosphorylation and dimer formation and directly binds to cysteineresidues in STAT3 (C259) and in the kinase domain of JAK1 (C1077).Biochemical assays have also established that the triterpenoids directlyinteract with critical cysteine residues on Keap1 (Dinkova-Kostova etal., 2005). Keapl is an actin-tethered protein that keeps thetranscription factor Nrf2 sequestered in the cytoplasm under normalconditions (Kobayashi & Yamamoto, 2005). Oxidative stress results inoxidation of the regulatory cysteine residues on Keap1 and causes therelease of Nrf2. Nrf2 then translocates to the nucleus and binds toantioxidant response elements (AREs) resulting in transcriptionalactivation of many antioxidant and anti-inflammatory genes. Anothertarget of the Keap1/Nrf2/ARE pathway is heme oxygenase 1 (HO-1). HO-1breaks down heme into bilirubin and carbon monoxide and plays manyantioxidant and anti-inflammatory roles (Maines & Gibbs, 2005). HO-1 hasrecently been shown to be potently induced by the triterpenoids (Liby etal., 2005), including RTA 402. RTA 402 and many structural analogs havealso been shown to be potent inducers of the expression of other Phase 2proteins (Yates et al., 2007).

RTA 402 is a potent inhibitor of NF-κB activation. Furthermore, RTA 402activates the Keap1/Nrf2/ARE pathway and induces expression of HO-1. Asdescribed below, RTA 402 has demonstrated activity in two animal modelsof AKI. Furthermore, reduced serum creatinine levels and improvement ofglomerular filtration have been observed in the majority of humanpatients that have been treated with RTA 402 (see Examples below).Significant improvements have now been observed in a Phase II study ofpatients with diabetic nephropathy. The findings indicate that RTA 402may be used to improve renal function in patients with diabeticnephropathy through suppression of renal inflammation and improvement ofglomerular filtration.

As noted above, both diabetes and essential hypertension are major riskfactors for the development of chronic kidney disease and, ultimately,renal failure. Both of these conditions, along with indicators ofsystemic cardiovascular disease such as hyperlipidemia, are frequentlypresent in the same patient, especially if that patient is clinicallyobese. Although the unifying factors are not completely understood,dysfunction of the vascular endothelium has been implicated as asignificant pathological factor in systemic cardiovascular disease,chronic kidney disease, and diabetes (see, e.g., Zoccali, 2006). Acuteor chronic oxidative stress in vascular endothelial cells has beenimplicated in the development of endothelial dysfunction, and isstrongly associated with chronic inflammatory processes. Therefore, anagent capable of relieving oxidative stress and concomitant inflammationin the vascular endothelium may alleviate dysfunction and restoreendothelial homeostasis. Without being bound by theory, compounds of theinvention, by stimulating Nrf2-regulated endogenous antioxidantmechanisms, have shown the highly unusual ability to improve parametersrelated to renal function (e.g., serum creatinine and estimatedglomerular filtration rate), glycemic control and insulin resistance(e.g., hemoglobin A1c), and systemic cardiovascular disease (e.g.,circulating endothelial cells) in patients having abnormal clinicalvalues for these parameters. Currently, combination therapy is typicallyrequired in such patients to achieve improvements in measures ofglycemic control and cardiovascular disease, including the use ofangiotensin-converting enzyme inhibitors or angiotensin II receptorblockers to alleviate hypertension and slow the progression of chronickidney disease. By achieving simultaneous and clinically meaningfulimprovements in all of these parameters, especially measures of renalfunction, compounds of the invention represent a significant improvementover currently available therapies. In some aspects, the compounds ofthe present invention may be used to treat a combination of the aboveconditions as a single therapy, or in combination with fewer additionaltherapies than would currently be used.

These findings also indicate that administration of RTA 402 may be usedto protect patients from kidney damage such as from exposure toradiocontrast agents, as in the case of radiocontrast-inducednephropathy (RCN), as well as in other contexts. In one aspect, thecompounds of this invention may be used to treat ischemia-reperfusion-and/or chemotherapy-induced acute renal injury. For example, the resultsshown in Examples 2 and 3 below demonstrate that RTA 402 is protectivein animal models of ischemia-reperfusion- and chemotherapy-induced acuterenal injury.

Serum creatinine has been measured in several animal models treated withRTA 402. Significant reductions of serum creatinine levels relative tobaseline levels or levels in control animals have been observed incynomolgus monkeys, beagle dogs, and Sprague-Dawley rats (FIGS. 3A-D).This effect has been observed in rats with both forms of RTA 402(crystalline and amorphous).

RTA 402 reduces serum creatinine in patients. For example, improvementswere observed in cancer patients receiving RTA 402. In humans,nephrotoxicity is a dose-limiting side-effect of treatment withcisplatin. Cisplatin-induced damage to the proximal tubules is thoughtto be mediated by increased inflammation, oxidative stress, andapoptosis (Yao et al., 2007). Serum creatinine has also been measured inpatients with chronic kidney disease (CKD) enrolled in an open labelPhase II clinical trial of RTA 402 (Example 6). This study was designedwith multiple endpoints, in categories of insulin resistance,endothelial dysfunction/CVD, and CKD, including measurements ofhemoglobin A1c (A1c), a widely used phase 3 endpoint for glycemiccontrol.

Alc is a minor component of hemoglobin to which glucose is bound. Alcalso is referred to as glycosylated or glucosylated hemoglobin. Alc maybe separated by charge and size from the other hemoglobin A componentsin blood using high performance liquid chromatography (HPLC). BecauseAlc is not affected by short-term fluctuations in blood glucoseconcentrations, for example, due to meals, blood can be drawn for Alctesting without regard to when food was eaten. In healthy, non-diabeticpatients the Alc level is less than 7% of total hemoglobin. The normalrange is 4-5.9%. In poorly controlled diabetes, it can be 8.0% or above.It has been demonstrated that the complications of diabetes can bedelayed or prevented if the Alc level can be kept close to 7%.

Recently approved agents typically only reduce Alc levels an amount of0.4 to 0.80 over six months of treatment, with 28 day improvementstypically smaller. The table below shows six-month Hemoglobin AlcReductions by two approved agents, sitagliptin and pramlintide acetate(Aschner et al., 2006; Goldstein et al., 2007; Pullman et al., 2006).

Duration of DM Mean Drug (years) Study Design A1c Change Sitagliptin 4.3+/−placebo with 8.0 −0.8 A1c ≧ 7.0 4.4 +/−metformin with 8.9 −0.7 A1c ≧7.5 6.1 pioglitazone +/− 8.1 −0.7 sitagliptin; A1c ≧ 7.0 Pramlintide 13+/−insulin 9.1 −0.4 acetate

In comparison, RTA 402 reduces Alc in 28 days in refractory diabetics ontop of standard of care. The treatment showed an intent-to-treatreduction of 0.34 (n=21) and an elevated baseline (≧7.0 at baseline)reduction of 0.50 (n=16). These results are presented in greater detailin the Examples section below. See also FIGS. 6 and 7.

In another aspect, the compounds of this invention may also be used toimprove insulin sensitivity and/or glycemic control. For example,hyperinsulinemic euglycemic clamp test results in the study detailed inExample 6 showed that treatment with RTA 402 improved glycemic control.The hyperinsulinemic euglycemic clamp test is a standard method forinvestigating and quantifying insulin sensitivity. It measures theamount of glucose necessary to compensate for an increased insulin levelwithout causing hypoglycemia (DeFronzo et al., 1979).

The typical procedure is as follows: Through a peripheral vein, insulinis infused at 10-120 mU per m² per minute. In order to compensate forthe insulin infusion, glucose 20% is infused to maintain blood sugarlevels between 5 and 5.5 mmol/liter. The rate of glucose infusion isdetermined by checking the blood sugar levels every 5 to 10 minutes.

Typically, low-dose insulin infusions are more useful for assessing theresponse of the liver, whereas high-dose insulin infusions are usefulfor assessing peripheral (i.e., muscle and fat) insulin action.

Results are typically evaluated as follows: The rate of glucose infusionduring the last 30 minutes of the test determines insulin sensitivity.If high levels (7.5 mg/min or higher) are required, the patient isinsulin-sensitive. Very low levels (4.0 mg/min or lower) indicate thatthe body is resistant to insulin action. Levels between 4.0 and 7.5mg/min may not be definitive and may suggest “impaired glucosetolerance,” an early sign of insulin resistance.

The methods of this invention may be used to improve renal function. Asshown in Example 6, treatment using RTA 402 has been shown to improvesix measures of renal function and status, including serum creatininebased eGFR, creatinine clearance, BUN, Cystatin C, Adiponectin, andAngiotensin II. RTA 402 was shown to increase GFR in a dose-dependentmanner and with high response rate (86%; n=22). As also shown in FIG. 9,the 28 day GFR improvements were reversible after the drug waswithdrawn.

In some embodiments, treatment methods of this invention result inimproved levels of Adiponectin and/or Angiotensin II. Adiponectin andAngiotensin II are typically elevated in DN patients and correlate withrenal disease severity. Adiponectin (also referred to as Acrp30, apM1)is a hormone known to modulate a number of metabolic processes,including glucose regulation and fatty acid catabolism. Adiponectin issecreted from adipose tissue into the bloodstream and is abundant inplasma relative to many other hormones. Levels of the hormone areinversely correlated with body fat percentage in adults, while theassociation in infants and young children is more unclear. The hormoneplays a role in the suppression of the metabolic derangements that mayresult in type 2 diabetes, obesity, atherosclerosis and non-alcoholicfatty liver disease (NAFLD). Adiponectin can be used to predictall-cause mortality and end stage renal disease in DN patients.

The compounds and methods of this invention may be used for treatingvarious aspects of cardiovascular disease (CVD). The treatment methodsof this invention have been found to reduce circulating endothelialcells (CECs) in human patients. CECs are markers of endothelialdysfunction and vascular injury. Endothelial dysfunction is a systemicinflammatory process that is linked to cardiovascular and end-organdamage. Elevated CECs typically correlate with the development,progression, and death from CVD. They also typically correlate withchronic kidney disease and decreased GFR. Historical normal levels are<5 cells/mL.

Typical features of endothelial dysfunction include the inability ofarteries and arterioles to dilate fully in response to an appropriatestimulus. This creates a detectable difference in subjects withendothelial dysfunction versus a normal, healthy endothelium. Such adifference can be tested by a variety of methods including iontophoresisof acetylcholine, intra-arterial administration of various vasoactiveagents, localised heating of the skin or temporary arterial occlusion byinflating a blood pressure cuff to high pressures. Testing can also takeplace in the coronary arteries themselves. These techniques are thoughtto stimulate the endothelium to release nitric oxide (NO) and possiblysome other agents, which diffuse into the surrounding vascular smoothmuscle causing vasodilation.

For example, according to the Phase II study results (Example 6),patients treated with RTA 402 for 28 days showed a reduction incardiovascular inflammatory markers in the form of a reduction in thenumber of circulating endothelial cells. The reduction in CECs for theintent-to-treat group (n=20) was 27%; the reduction for the elevatedbaseline group (n=14) was 40% (p=0.02) and nine of those patients showeda normal level for CECs post-treatment. These results are consistentwith a reversal of endothelial dysfunction.

The treatment methods of this invention have been found to reduce matrixmetallopeptidase 9 (MMP-9), soluble adhesion molecules and tumornecrosis factor (TNFα) in most patients. High levels of these typicallycorrelate with poor cardiovascular outcomes.

VI. Pharmaceutical Formulations and Routes of Administration

Administration of the compounds of the present invention to a patientwill follow general protocols for the administration of pharmaceuticals,taking into account the toxicity, if any, of the drug. It is expectedthat the treatment cycles would be repeated as necessary.

The compounds of the present invention may be administered by a varietyof methods, e.g., orally or by injection (e.g. subcutaneous,intravenous, intraperitoneal, etc.). Depending on the route ofadministration, the active compounds may be coated by a material toprotect the compound from the action of acids and other naturalconditions which may inactivate the compound. They may also beadministered by continuous perfusion/infusion of a disease or woundsite. Specific examples of formulations, including a polymer-baseddispersion of CDDO-Me that showed improved oral bioavailability, areprovided in U.S. application Ser. No. 12/191,176, filed Aug. 13, 2008,which is incorporated herein by reference in its entirety. It will berecognized by those skilled in the art that other methods of manufacturemay be used to produce dispersions of the present invention withequivalent properties and utility (see Repka et al., 2002 and referencescited therein). Such alternative methods include but are not limited tosolvent evaporation, extrusion, such as hot melt extrusion, and othertechniques.

To administer the therapeutic compound by other than parenteraladministration, it may be necessary to coat the compound with, orco-administer the compound with, a material to prevent its inactivation.For example, the therapeutic compound may be administered to a patientin an appropriate carrier, for example, liposomes, or a diluent.Pharmaceutically acceptable diluents include saline and aqueous buffersolutions. Liposomes include water-in-oil-in-water CGF emulsions as wellas conventional liposomes (Strejan et al., 1984).

The therapeutic compound may also be administered parenterally,intraperitoneally, intraspinally, or intracerebrally. Dispersions may beprepared in, e.g., glycerol, liquid polyethylene glycols, mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations may contain a preservative to prevent the growth ofmicroorganisms.

Pharmaceutical compositions suitable for injectable use include sterileaqueous solutions (where water soluble) or dispersions and sterilepowders for the extemporaneous preparation of sterile injectablesolutions or dispersion. In all cases, the composition must be sterileand must be fluid to the extent that easy syringability exists. It mustbe stable under the conditions of manufacture and storage and must bepreserved against the contaminating action of microorganisms such asbacteria and fungi. The carrier may be a solvent or dispersion mediumcontaining, for example, water, ethanol, polyol (such as, glycerol,propylene glycol, and liquid polyethylene glycol, and the like),suitable mixtures thereof, and vegetable oils. The proper fluidity canbe maintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case of dispersionand by the use of surfactants. Prevention of the action ofmicroorganisms can be achieved by various antibacterial and antifungalagents, for example, parabens, chlorobutanol, phenol, ascorbic acid,thimerosal, and the like. In many cases, it will be preferable toinclude isotonic agents, for example, sugars, sodium chloride, orpolyalcohols such as mannitol and sorbitol, in the composition.Prolonged absorption of the injectable compositions can be brought aboutby including in the composition an agent which delays absorption, forexample, aluminum monostearate or gelatin.

Sterile injectable solutions can be prepared by incorporating thetherapeutic compound in the required amount in an appropriate solventwith one or a combination of ingredients enumerated above, as required,followed by filtered sterilization. Generally, dispersions are preparedby incorporating the therapeutic compound into a sterile carrier whichcontains a basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient (i.e., the therapeutic compound) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The therapeutic compound can be orally administered, for example, withan inert diluent or an assimilable edible carrier. The therapeuticcompound and other ingredients may also be enclosed in a hard or softshell gelatin capsule, compressed into tablets, or incorporated directlyinto the subject's diet. For oral therapeutic administration, thetherapeutic compound may be incorporated with excipients and used in theform of ingestible tablets, buccal tablets, troches, capsules, elixirs,suspensions, syrups, wafers, and the like. The percentage of thetherapeutic compound in the compositions and preparations may, ofcourse, be varied. The amount of the therapeutic compound in suchtherapeutically useful compositions is such that a suitable dosage willbe obtained.

It is especially advantageous to formulate parenteral compositions indosage unit form for ease of administration and uniformity of dosage.Dosage unit form as used herein refers to physically discrete unitssuited as unitary dosages for the subjects to be treated; each unitcontaining a predetermined quantity of therapeutic compound calculatedto produce the desired therapeutic effect in association with therequired pharmaceutical carrier. The specification for the dosage unitforms of the invention are dictated by and directly dependent on (a) theunique characteristics of the therapeutic compound and the particulartherapeutic effect to be achieved, and (b) the limitations inherent inthe art of compounding such a therapeutic compound for the treatment ofa selected condition in a patient.

The therapeutic compound may also be administered topically to the skin,eye, or mucosa. Alternatively, if local delivery to the lungs is desiredthe therapeutic compound may be administered by inhalation in adry-powder or aerosol formulation.

The actual dosage amount of a compound of the present invention orcomposition comprising a compound of the present invention administeredto a subject may be determined by physical and physiological factorssuch as age, sex, body weight, severity of condition, the type ofdisease being treated, previous or concurrent therapeutic interventions,idiopathy of the subject and on the route of administration. Thesefactors may be determined by a skilled artisan. The practitionerresponsible for administration will typically determine theconcentration of active ingredient(s) in a composition and appropriatedose(s) for the individual subject. The dosage may be adjusted by theindividual physician in the event of any complication.

In some embodiments, the pharmaceutically effective amount is a dailydose from about 0.1 mg to about 500 mg of the compound. In somevariations, the daily dose is from about 1 mg to about 300 mg of thecompound. In some variations, the daily dose is from about 10 mg toabout 200 mg of the compound. In some variations, the daily dose isabout 25 mg of the compound. In other variations, the daily dose isabout 75 mg of the compound. In still other variations, the daily doseis about 150 mg of the compound. In further variations, the daily doseis from about 0.1 mg to about 30 mg of the compound. In some variations,the daily dose is from about 0.5 mg to about 20 mg of the compound. Insome variations, the daily dose is from about 1 mg to about 15 mg of thecompound. In some variations, the daily dose is from about 1 mg to about10 mg of the compound. In some variations, the daily dose is from about1 mg to about 5 mg of the compound.

In some embodiments, the pharmaceutically effective amount is a dailydose is 0.01-25 mg of compound per kg of body weight. In somevariations, the daily dose is 0.05-20 mg of compound per kg of bodyweight. In some variations, the daily dose is 0.1-10 mg of compound perkg of body weight. In some variations, the daily dose is 0.1-5 mg ofcompound per kg of body weight. In some variations, the daily dose is0.1-2.5 mg of compound per kg of body weight.

In some embodiments, the pharmaceutically effective amount is a dailydose is of 0.1-1000 mg of compound per kg of body weight. In somevariations, the daily dose is 0.15-20 mg of compound per kg of bodyweight. In some variations, the daily dose is 0.20-10 mg of compound perkg of body weight. In some variations, the daily dose is 0.40-3 mg ofcompound per kg of body weight. In some variations, the daily dose is0.50-9 mg of compound per kg of body weight. In some variations, thedaily dose is 0.60-8 mg of compound per kg of body weight. In somevariations, the daily dose is 0.70-7 mg of compound per kg of bodyweight. In some variations, the daily dose is 0.80-6 mg of compound perkg of body weight. In some variations, the daily dose is 0.90-5 mg ofcompound per kg of body weight. In some variations, the daily dose isfrom about 1 mg to about 5 mg of compound per kg of body weight.

An effective amount typically will vary from about 0.001 mg/kg to about1,000 mg/kg, from about 0.01 mg/kg to about 750 mg/kg, from about 0.1mg/kg to about 500 mg/kg, from about 0.2 mg/kg to about 250 mg/kg, fromabout 0.3 mg/kg to about 150 mg/kg, from about 0.3 mg/kg to about 100mg/kg, from about 0.4 mg/kg to about 75 mg/kg, from about 0.5 mg/kg toabout 50 mg/kg, from about 0.6 mg/kg to about 30 mg/kg, from about 0.7mg/kg to about 25 mg/kg, from about 0.8 mg/kg to about 15 mg/kg, fromabout 0.9 mg/kg to about 10 mg/kg, from about 1 mg/kg to about 5 mg/kg,from about 100 mg/kg to about 500 mg/kg, from about 1.0 mg/kg to about250 mg/kg, or from about 10.0 mg/kg to about 150 mg/kg, in one or moredose administrations daily, for one or several days (depending, ofcourse, of the mode of administration and the factors discussed above).Other suitable dose ranges include 1 mg to 10,000 mg per day, 100 mg to10,000 mg per day, 500 mg to 10,000 mg per day, and 500 mg to 1,000 mgper day. In some particular embodiments, the amount is less than 10,000mg per day with a range, for example, of 750 mg to 9,000 mg per day.

The effective amount may be less than 1 mg/kg/day, less than 500mg/kg/day, less than 250 mg/kg/day, less than 100 mg/kg/day, less than50 mg/kg/day, less than 25 mg/kg/day, less than 10 mg/kg/day, or lessthan 5 mg/kg/day. It may alternatively be in the range of 1 mg/kg/day to200 mg/kg/day. For example, regarding treatment of diabetic patients,the unit dosage may be an amount that reduces blood glucose by at least40% as compared to an untreated subject. In another embodiment, the unitdosage is an amount that reduces blood glucose to a level that is within+10% of the blood glucose level of a non-diabetic subject.

In other non-limiting examples, a dose may also comprise from about 1micro-gram/kg/body weight, about 5 microgram/kg/body weight, about 10microgram/kg/body weight, about 50 microgram/kg/body weight, about 100microgram/kg/body weight, about 200 microgram/kg/body weight, about 350microgram/kg/body weight, about 500 microgram/kg/body weight, about 1milligram/kg/body weight, about 5 milli-gram/kg/body weight, about 10milligram/kg/body weight, about 50 milligram/kg/body weight, about 100milligram/kg/body weight, about 200 milligram/kg/body weight, about 350milligram/kg/body weight, about 500 milligram/kg/body weight, to about1000 mg/kg/body weight or more per administration, and any rangederivable therein. In non-limiting examples of a derivable range fromthe numbers listed herein, a range of about 1 mg/kg/body weight to about5 mg/kg/body weight, a range of about 5 mg/kg/body weight to about 100mg/kg/body weight, about 5 microgram/kg/body weight to about 500milligram/kg/body weight, etc., can be administered, based on thenumbers described above.

In certain embodiments, a pharmaceutical composition of the presentinvention may comprise, for example, at least about 0.1% of a compoundof the present invention. In other embodiments, the compound of thepresent invention may comprise between about 2% to about 75% of theweight of the unit, or between about 25% to about 60%, for example, andany range derivable therein.

Single or multiple doses of the agents are contemplated. Desired timeintervals for delivery of multiple doses can be determined by one ofordinary skill in the art employing no more than routineexperimentation. As an example, subjects may be administered two dosesdaily at approximately 12 hour intervals. In some embodiments, the agentis administered once a day.

The agent(s) may be administered on a routine schedule. As used herein aroutine schedule refers to a predetermined designated period of time.The routine schedule may encompass periods of time which are identicalor which differ in length, as long as the schedule is predetermined. Forinstance, the routine schedule may involve administration twice a day,every day, every two days, every three days, every four days, every fivedays, every six days, a weekly basis, a monthly basis or any set numberof days or weeks there-between. Alternatively, the predetermined routineschedule may involve administration on a twice daily basis for the firstweek, followed by a daily basis for several months, etc. In otherembodiments, the invention provides that the agent(s) may taken orallyand that the timing of which is or is not dependent upon food intake.Thus, for example, the agent can be taken every morning and/or everyevening, regardless of when the subject has eaten or will eat.

Non-limiting specific formulations include CDDO-Me polymer dispersions(see U.S. application Ser. No. 12/191,176, filed Aug. 13, 2008, which isincorporated herein by reference). Some of the formulations reportedtherein exhibited higher bioavailability than either the micronized FormA or nanocrystalline Form A formulations. Additionally, the polymerdispersion based formulations demonstrated further surprisingimprovements in oral bioavailability relative to the micronized Form Bformulations. For example, the methacrylic acid copolymer, Type C andHPMC-P formulations showed the greatest bioavailability in the subjectmonkeys.

VII. Combination Therapy

In addition to being used as a monotherapy, the compounds of the presentinvention may also find use in combination therapies. Effectivecombination therapy may be achieved with a single composition orpharmacological formulation that includes both agents, or with twodistinct compositions or formulations, administered at the same time,wherein one composition includes a compound of this invention, and theother includes the second agent(s). Alternatively, the therapy mayprecede or follow the other agent treatment by intervals ranging fromminutes to months.

Various combinations may be employed, such as when a compound of thepresent invention is “A” and “B” represents a secondary agent,non-limiting examples of which are described below:

A/B/A B/A/B B/B/A A/A/B A/B/B B/A/A A/B/B/B B/A/B/BB/B/B/A   B/B/A/B   A/A/B/B   A/B/A/B  A/B/B/A  B/B/A/AB/A/B/A   B/A/A/B   A/A/A/B   B/A/A/A  A/B/A/A  A/A/B/A

It is contemplated that other anti-inflammatory agents may be used inconjunction with the treatments of the current invention. For example,other COX inhibitors may be used, including arylcarboxylic acids(salicylic acid, acetylsalicylic acid, diflunisal, choline magnesiumtrisalicylate, salicylate, benorylate, flufenamic acid, mefenamic acid,meclofenamic acid and triflumic acid), arylalkanoic acids (diclofenac,fenclofenac, alclofenac, fentiazac, ibuprofen, flurbiprofen, ketoprofen,naproxen, fenoprofen, fenbufen, suprofen, indoprofen, tiaprofenic acid,benoxaprofen, pirprofen, tolmetin, zomepirac, clopinac, indomethacin andsulindac) and enolic acids (phenylbutazone, oxyphenbutazone,azapropazone, feprazone, piroxicam, and isoxicam. See also U.S. Pat. No.6,025,395, which is incorporated herein by reference.

Dietary and nutritional supplements with reported benefits for treatmentor prevention of Parkinson's, Alzheimer's, multiple sclerosis,amyotrophic lateral sclerosis, rheumatoid arthritis, inflammatory boweldisease, and all other diseases whose pathogenesis is believed toinvolve excessive production of either nitric oxide (NO) orprostaglandins, such as acetyl-L-carnitine, octacosanol, eveningprimrose oil, vitamin B6, tyrosine, phenylalanine, vitamin C, L-dopa, ora combination of several antioxidants may be used in conjunction withthe compounds of the current invention.

Other particular secondary therapies include immunosuppressants (fortransplants and autoimmune-related RKD), anti-hypertensive drugs (forhigh blood pressure-related RKD, e.g., angiotensin-converting enzymeinhibitors and angiotensin receptor blockers), insulin (for diabeticRKD), lipid/cholesterol-lowering agents (e.g., HMG-CoA reductaseinhibitors such as atorvastatin or simvastatin), treatments forhyperphosphatemia or hyperparathyroidism associated with CKD (e.g.,sevelamer acetate, cinacalcet), dialysis, and dietary restrictions(e.g., protein, salt, fluid, postassium, phosphorus).

VIII. Examples

The following examples are included to demonstrate preferred embodimentsof the invention. It should be appreciated by those of skill in the artthat the techniques disclosed in the examples which follow representtechniques discovered by the inventor to function well in the practiceof the invention, and thus can be considered to constitute preferredmodes for its practice. However, those of skill in the art should, inlight of the present disclosure, appreciate that many changes can bemade in the specific embodiments which are disclosed and still obtain alike or similar result without departing from the spirit and scope ofthe invention.

Example 1 Materials and Methods

Chemicals. Triterpenoids were synthesized as previously described inHonda et al. (1998), Honda et al. (2000b), Honda et al. (2002) and Yateset al. (2007), which are all incorporated herein by reference.

Example 2 Mouse Ischemia-Reperfusion Results

In a mouse model of ischemic acute renal failure, the renal artery isclamped for approximately twenty minutes. After this time, the clamp isremoved and the kidney is reperfused with blood. Ischemia-reperfusionresults in renal damage and decreased renal function which can beassessed by blood urea nitrogen (BUN) levels, which become elevatedfollowing renal damage. As shown in FIGS. 1 a-d, surgically-inducedischemia-reperfusion increased BUN levels by approximately 2-fold.However, in animals treated with 2 mg/kg RTA 402 orally once dailybeginning two days prior to the surgery, the BUN levels weresignificantly reduced (p<0.01) relative to vehicle-treated animals andwere similar to the levels in animals that underwent sham surgeries(FIGS. 1 a-c). Histological measures of kidney damage and inflammationwere also significantly improved by treatment with RTA 402 (FIG. 1 d).These data indicate that RTA 402 is protective againstischemia-reperfusion induced tissue damage.

Example 3 Rat Chemotherapy-Induced Acute Renal Injury Results

In another model of acute renal injury, rats were injected intravenouslywith the antineoplastic agent cisplatin. In humans, nephrotoxicity is adose-limiting side effect of treatment with cisplatin. Cisplatin-induceddamage to the proximal tubules is thought to be mediated by increasedinflammation, oxidative stress, and apoptosis (Yao et al., 2007). Ratstreated with a single dose of cisplatin at 6 mg/kg developed renalinsufficiency as measured by increased blood levels of creatinine andBUN. Treatment with 10 mg/kg RTA 402 by oral gavage beginning one dayprior to treatment with cisplatin and continuing every day significantlyreduced blood levels of creatinine and BUN (FIGS. 2 a-b). Histologicalevaluation of the kidneys demonstrated an improvement in the extent ofproximal tubule damage in RTA 402-treated animals compared tovehicle-treated animals (FIG. 2 c).

Example 4 Reduction of Serum Creatinine Levels in Several Species

Serum creatinine has been measured in several animal species treatedwith RTA 402 in the course of toxicology studies. Significant reductionsof serum creatinine levels relative to baseline levels or levels incontrol animals have been observed in cynomolgus monkeys, beagle dogs,and Sprague-Dawley rats (FIGS. 3 a-d). This effect has been observed inrats with crystalline and amorphous forms of RTA 402.

Example 5 Reduced Serum Creatinine and Increased eGFR in Cancer Patients

Serum creatinine has also been measured in patients with cancer enrolledin a Phase I clinical trial of RTA 402. These patients received RTA 402once daily at doses from 5 to 1,300 mg/day for a total of twenty-onedays every 28 days. A reduction in serum creatinine by greater than 15%was observed as early as eight days following treatment initiation andpersisted through the end of the cycle (FIG. 4A). This reduction wasmaintained in those patients that received six or more cycles oftreatment with RTA 402. A subset of patients with pre-existing renaldamage (baseline serum creatinine levels of at least 1.5 mg/dl) also hadsignificant reductions in serum creatinine levels following treatmentwith RTA 402. In these patients, serum creatinine levels decreasedprogressively throughout the cycle such that the Day 21 levels wereapproximately 25% lower than baseline levels (FIG. 4A). These resultscan be summarized as shown in the table below.

Sub-set with elevated baseline serum All patients creatinine levelsNumber of patients who received drug 45 8 for at least 3 weeks % ofPatients with Decrease on Day 21   87%   100% % Serum CreatinineDecrease from −18.3% −24.5% Baseline p-value (Baseline versus Day 21)0.001 0.0007

The estimated glomerular filtration rate (eGFR) significantly improvedin the patients treated with RTA 402 (FIG. 4B).

FIG. 5 shows the results following at least six months of RTA 402treatment in eleven cancer patients, showing that eGFR improved in anapproximately continuous manner. Some of these patients were enrolled inthe Phase I study, whereas others were enrolled in a study of RTA 402(in combination with gemcitabine) in patients with pancreatic cancer.The results can be summarized as shown in Table 2, below.

TABLE 2 eGFR in Patients Receiving RTA 402 for 6 Cycles. Solid TumorStudy Pancreatic Study Pt ID: 402 406 408 409 410 421 427 1001 1104 11051106 Dose (mg): 5 80 150 150/300 300/600 1300/900 1300 150 300/150 300300 Cycle BL 109.7 94.2 73.2 48.4 49.9 52.5 70.1 68.8 67.3 82.4 89.0(each cycle 1 109.7 125.9 82.1 62.6 69.6 58.6 101.3 78.9 95.7 106.6106.3 is 28 days) 2 109.7 107.9 77.4 62.6 63.4 66.2 78.3 109.9 71.6 89.3106.3 3 95.7 107.9 69.4 62.6 63.4 75.8 88.4 135.7 141.2 106.6 106.3 495.7 125.9 77.4 57.0 69.6 N/A 101.3 175.5 95.7 106.6 131.2 5 109.7 107.977.4 69.2 63.4 88.4 101.3 175.5 114.4 131.6 131.2 6 95.7 125.9 87.4 69.269.6 75.8 101.3 135.7 114.4 170.3 131.2

Example 6 Phase 2 Study in Patients with Diabetic Nephropathy

Serum creatinine has also been measured in patients with chronic kidneydisease (CKD) enrolled in an open label Phase II clinical trial of RTA402. These patients received RTA 402 once daily at three dose levels, 25mg, 75 mg and 150 mg, for a total of 28 days.

The study was designed with multiple endpoints, in categories of insulinresistance, endothelial dysfunction/CVD, and CKD. These can besummarized as follows:

Endothelial Insulin Dysfunction/ Chronic Kidney Resistance/DiabetesCardiovascular Disease Hgb A1c CECs GFR GDR/Euglycemic C-ReactiveProtein Serum Creatinine Clamp (CRP) Glucose E-Selectin CreatinineClearance VCAM Cystatin C Cytokines Adiponectin Angiotensin II

A primary outcome measure for this study is determining the effects ofRTA 402 administered orally at the three dose strengths on theglomerular filtration rate (as estimated by the MDRD formula) inpatients with diabetic nephropathy.

Secondary outcome measures include: (1) an evaluation of the safety andtolerability of oral RTA 402 administered orally at the three differentdoses, in this patient population; (2) an evaluation of the effects ofRTA 402 administered orally at the three dose strengths on the serumcreatinine level, creatinine clearance, and urine albumin/creatinineratio in patients with diabetic nephropathy; (3) an evaluation of theeffects of RTA 402 administered orally at the three dose strengths onhemoglobin Alc in all patients enrolled and on insulin response by thehyperinsulinemic euglycemic clamp test in patients enrolled at only oneof the study centers; (4) an evaluation of the effects of RTA 402 at thethree different doses on a panel of markers of inflammation, renalinjury, oxidative stress, and endothelial cell dysfunction.

The patient population selected for this study all had type 2 diabeteswith CKD. Most had been diagnosed with poor glycemic control for twodecades. CKD was established through elevated serum creatinine (SCr)levels. Most of the patients had been diagnosed with cardiovasculardisease (CVD) and most were receiving standard of care (SOC) treatmentfor diabetes, CKD and CVD, (e.g., insulin, ACEI/ARB, β-blocker,diuretic, and statin). The baseline demographic can be summarized asfollows:

Age 59 Diabetes Duration (yrs) 15.4 Diabetic Nephropathy 100%  Non-renalDiabetic Complications¹ 100%  Hypertensive 100%  Hgb A1c(%) 7.9%  FailedOral Antihyperglycemics 90% ACEI/ARB Use 80% Statin Use 50% ¹Includesneuropathy and retinopathy All values represent the mean; n = 10; 1^(st)10 patients to complete study

The patient inclusion criteria were as follows: (1) diagnosis of type 2diabetes; (2) serum creatinine in women 1.3-3.0 mg/dL (115-265 μmol/L),inclusive, and in men 1.5-3.0 mg/dL (133-265 μmol/L), inclusive; (3)patient must agree to practice effective contraception; (4) patient musthave a negative urine pregnancy test within 72 hours prior to the firstdose of study medication; (5) patient is willing and able to cooperatewith all aspects of the protocol and is able to communicate effectively;(6) patient is willing and able to provide written informed consent toparticipate in this clinical study.

The patient exclusion criteria were the following: (1) patients havingtype 1 (insulin-dependent; juvenile onset) diabetes; (2) patients withknown non-diabetic renal disease (nephrosclerosis superimposed ondiabetic nephropathy acceptable), or with renal allograft; (3) patientshaving cardiovascular disease as follows: unstable angina pectoriswithin 3 months of study entry; myocardial infarction, coronary arterybypass graft surgery, or percutaneous transluminal coronaryangioplasty/stent within 3 months of study entry; transient ischemicattack within 3 months of study entry; cerebrovascular accident within 3months of study entry; obstructive valvular heart disease orhypertrophic cardiomyopathy; second or third degree atrioventricularblock not successfully treated with a pacemaker; (4) patients with needfor chronic (>2 weeks) immunosuppressive therapy, includingcorticosteroids (excluding inhaled or nasal steroids) within 3 months ofstudy entry; (5) patients with evidence of hepatic dysfunction includingtotal bilirubin>1.5 mg/dL (>26 micromole/L) or liver transaminase(aspartate aminotransferase [AST] or alanine transferase [ALT])>1.5times upper limit of normal; (6) if female, patient is pregnant, nursingor planning a pregnancy; (7) patients with any concurrent clinicalconditions that in the judgment of the investigator could eitherpotentially pose a health risk to the patient while involved in thestudy or could potentially influence the study outcome; (8) patientshaving known hypersensitivity to any component of the study drug; (9)patients having known allergy to iodine; (10) patients having undergonediagnostic or intervention procedure requiring a contrast agent withinthe last 30 days prior to entry into the study; (11) patients withchange or dose-adjustment in any of the following medications: ACEinhibitors, angiotensin II blockers, non-steroidal anti inflammatorydrugs (NSAIDs), or COX-2 inhibitors within 3 months; otheranti-hypertensive, and other anti-diabetic medications within 6 weeksprior to entry into the study; (12) patients having a history of drug oralcohol abuse or having positive test results for any drug of abuse(positive urine drug test and/or alcohol breathalyzer test); (13)patients having participated in another clinical study involvinginvestigational or marketed products within 30 days prior to entry intothe study or would concomitantly participate in such a study; (14)patients unable to communicate or cooperate with the Investigator due tolanguage problems, poor mental development or impaired cerebralfunction.

As of the end of September 2008, there were 32 of 60 patients enrolledin this study. All but one patient was receiving insulin andstandard-of-care oral antihyperglycemics.

Treatment with RTA 402 was observed to reduce hemoglobin % Alc in 28days in refractory diabetics on top of standard of care. The treatmentshowed an intent-to-treat reduction of approximately 0.25 (n=56) and anelevated baseline (>7.0 at baseline) reduction of 0.50 (n=35).Hemoglobin % Alc reduction as a function of baseline severity is shownin FIG. 6, and reduction as a function of dosage is shown in FIG. 7.Patients with advanced (Stage 4) renal disease (GFR from 15-29 ml/min)showed a mean % Alc reduction of approximately 0.77. All reductions werestatistically significant.

Hyperinsulinemic euglycemic clamp test results showed that the 28 daytreatment also improved glycemic control and insulin sensitivity in thepatients, as measured by glucose disposal rate (GDR). Patients exhibitedimprovements in GDR after the 28 day treatment, with more severelyimpaired patients (GDR<4) showing statistically significant improvements(p≦0.02). The hyperinsulinemic euglycemic clamp test was performed atBaseline (Day −1) and at the end of the study on Day 28. The testmeasures the rate of glucose infusion (GINF) necessary to compensate foran increased insulin level without causing hypoglycemia; this value isused to derive the GDR.

In short, the hyperinsulinemic euglycemic clamp test takes about 2hours. Through a peripheral vein, insulin is infused at 10-120 mU per m²per minute. In order to compensate for the insulin infusion, glucose 20%is infused to maintain blood sugar levels between 5 and 5.5 mmol/L. Therate of glucose infusion is determined by checking the blood sugarlevels every 5 to 10 minutes. The rate of glucose infusion during thelast 30 minutes of the test is used to determine insulin sensitivity asdetermined by the glucose metabolism rate (M) in mg/kg/min.

The following protocol guidelines are in place for the hyperinsulinemiceuglycemic clamp test:

-   -   1) Subject to fast 8-10 hours prior to the clamp procedure.    -   2) The morning of the clamp measure vital signs and weight.    -   3) Start a retrograde line in one hand with 1¼″, 18-20 gauge        catheter for drawing samples.    -   4) Prepare IV tubing with 2 three-way stop cocks and j-loop        extension tubing. Spike tubing to a liter bag of 0.9% NaCl to        run at KVO (keep vein open, about 10 cc/hr) until the start of        the procedure.    -   5) Apply a heating pad covered in a pillow case with a pad        separating the heating pad from the subject's hand. (Enables the        collection of shunted arterialized blood from venous        catheterization)    -   6) Monitor the temperature (approximately 150° F./65° C.)        generated by the heating pad before and during the clamp, to        maintain arterialization.    -   7) Start another line opposite the draw side in the distal        forearm with 1¼″, 18-20 gauge catheter for the infusion line.        Prepare IV tubing with 2 three-way stop cocks.    -   8) Hang a 500 ml bag of 20% dextrose and attach to port on the        infusion side    -   9) Prepare the insulin infusion        -   a. Remove 53 cc (50 cc of overfill) of saline from a 500 cc            bag of 0.9% NaCl and discard        -   b. Draw 8 cc of blood from subject using sterile technique            and inject into a tiger top tube        -   c. Centrifuge the tiger top tube. Withdraw 2 cc of serum and            inject into the 500 cc bag of 0.9% NaCl        -   d. Add 100 units of insulin to the bag with the serum and            mix well (0.2 U insulin/ml)        -   e. Connect IV tubing with duo-vent spike into the 0.9% NaCl            bag        -   f. Place on Baxter pump    -   10) Time and draw all basal blood samples (Baseline fasting        blood glucose values will be obtained prior to beginning the        insulin prime).    -   11) Perform insulin infusion rate calculations for a priming        dose and 60 mU/m² insulin infusion. This background insulin is        to suppress endogenous hepatic glucose production. Lean subjects        can be suppressed with 40 mU/m 2; obese, insulin resistant        subjects require 80 mU/m². 60 mU/m² should be sufficient to        suppress the suggested study population with a BMI of 27-40        kg/m². The suggested 60 mU/m² insulin infusion may need to be        adjusted if the BMI is amended.    -   12) 0.5 mL samples will be drawn every five minutes and the        readings from the YSI Blood Glucose Analyzer will be used to        determine/adjust the glucose infusion rate (mg/kg/min). Any        additional laboratory tests required by the protocol will be in        addition to the blood volume. The clamp will last 120 minutes        which is believed to be a sufficient duration for determining        insulin sensitivity.    -   13) Label and save all YSI printouts for source documents.    -   14) The glucose infusion rates from the last 30 minutes of the        euglycemic clamp will be adjusted using space correction. This        will be used to determine the glucose metabolism rate (M        mg/kg/min), which represents the subject's sensitivity to        insulin.

As shown in FIG. 8, RTA 402 reduces circulating endothelial cells(CECs). The mean number of CECs in cells/mL is shown for intent-to-treat(ITT) and elevated baseline groups, both before and after the 28 day RTAtreatment. The reduction for the Intent-to-treat group was approximately20%, and the reduction in the elevated baseline group (>5 CECs/ml) wasapproximately 33%. The fraction of iNOS-positive CECs was reducedapproximately 29%. Normalization of CEC values (<5 cells/mL) wasobserved in 11 out of the 19 patients with elevated baseline.

CECs were isolated from whole blood by using CD146 Ab (an antibody tothe CD146 antigen that is expressed on endothelial cells andleukocytes). After CEC isolation, a FITC (fluorescein isothiocyanate)conjugated CD105 Ab (a specific antibody for endothelial cells) is usedto identify CECs using the CellSearch™ system. A fluorescent conjugateof CD45 Ab was added to stain the leukocytes, and these were then gatedout. For a general overview of this method, see Blann et al., (2005),which is incorporated herein by reference in its entirety. CEC sampleswere also assessed for the presence of iNOS by immunostaining. Treatmentwith RTA 402 reduced iNOS-positive CECs by approximately 29%, furtherindicating that RTA 402 reduces inflammation in endothelial cells.

RTA 402 was shown to improve significantly eight measures of renalfunction and status, including serum creatinine based eGFR (FIG. 9),creatinine clearance, BUN (FIG. 11A), serum phosphorus (FIG. 11B), serumuric acid (FIG. 1 IC), Cystatin C, Adiponectin (FIG. 10A), andAngiotensin II (FIG. 10B). Adiponectin predicts all-cause mortality andend stage renal disease in DN patients. Adiponectin and Angiotensin II,which are elevated in DN patients, correlate with renal disease severity(FIGS. 10A-B). Effects on BUN, phosphorus, and uric acid are shown inFIGS. 11A-C.

Patients treated with higher doses (75 or 150 mg) of RTA 402 showedmodest elevations (approximately 20 to 25%) in proteinuria. This isconsistent with studies indicating that better GFR performancecorrelates with increased proteinuria. For example, in a long-termclinical study of more than 25,000 patients, treatment with ramipril (anACE inhibitor) slowed the rate of eGFR decline more effectively thaneither telmisartan (an angiotensin receptor blocker) or the combinationof ramipril and telmisartan (Mann et al., 2008). Conversely, proteinuriaincreased more in the ramipril group than in the other two groups. Majorrenal outcomes were also better with either drug alone than withcombination therapy, although proteinuria increased least in thecombination therapy group. Other studies have shown that drugs thatreduce GFR, such as ACE-inhibitors, also reduce proteinuria (Lozano etal., 2001; Sengul et al., 2006). Other studies have shown that drugsthat acutely increase GFR, such as certain calcium channel blockers,increase proteinuria up to 60% during short-term dosing (Agodoa et al.,2001; Viberti et al., 2002).

All of the compositions and/or methods disclosed and claimed herein canbe made and executed without undue experimentation in light of thepresent disclosure. While the compositions and methods of this inventionhave been described in terms of preferred embodiments, it will beapparent to those of skill in the art that variations may be applied tothe compositions and/or methods and in the steps or in the sequence ofsteps of the method described herein without departing from the concept,spirit and scope of the invention. More specifically, it will beapparent that certain agents which are both chemically andphysiologically related may be substituted for the agents describedherein while the same or similar results would be achieved. All suchsimilar substitutes and modifications apparent to those skilled in theart are deemed to be within the spirit, scope and concept of theinvention as defined by the appended claims.

IX. References

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1-157. (canceled)
 158. A method of improving kidney function in asubject in need thereof comprising administering to the subject acompound of the formula,

in an amount sufficient to improve kidney function.
 159. The method ofclaim 158, where the subject has chronic kidney disease (CKD) orexhibits one or more symptoms of CKD.
 160. The method of claim 159,where the subject has been identified as having CKD.
 161. The method ofclaim 159, where the CKD is characterized by a serum creatinine level of1.3-3.0 mg/DL where the subject is a human female or a serum creatininelevel of 1.5-3.0 mg/DL where the subject is a human male.
 162. Themethod of claim 159, where the CKD is stage
 4. 163. The method of claim158, where the subject has diabetic nephropathy (DN) or exhibits one ormore symptoms of DN.
 164. The method of claim 163, where the subject hasbeen identified as having DN.
 165. The method of claim 158, where theadministering results in an improvement in estimated glomerularfiltration rate (eGFR) of the subject.
 166. The method of claim 165,where the administering reduces the level of serum creatinine in thesubject.
 167. The method of claim 166, where the level of serumcreatinine in the blood of the subject has been measured or will bemeasured.
 168. The method of claim 158, where the level of blood ureanitrogen (BUN) in the subject has been measured or will be measured.169. The method of claim 158, where the level of Adiponectin in theblood of the subject has been measured or will be measured.
 170. Themethod of claim 158, where the level of Angiotensin II in the subjecthas been measured or will be measured.
 171. The method of claim 158,where the subject has insulin resistance or exhibits one or moresymptoms of insulin resistance.
 172. The method of claim 171, where thesubject has been identified as having insulin resistance.
 173. Themethod of claim 171, where the level of hemoglobin Alc in the subjecthas been measured or will be measured.
 174. The method of claim 171,where a blood sugar level of the subject has been measured or will bemeasured.
 175. The method of claim 171, where the administering reducesthe level of hemoglobin Alc or fasting blood glucose in the subject.176. The method of claim 174, where a fasting glucose level of thesubject has been measured or will be measured.
 177. The method of claim171, where the insulin sensitivity of the subject has been measured orwill be measured by a hyperinsulinemic euglycemic clamp test.
 178. Themethod of claim 171, where a glucose disposal rate (GDR) in the subjecthas been measured or will be measured.
 179. The method of claim 158,where the subject has cardiovascular disease (CVD) or exhibits one ormore symptoms of CVD.
 180. The method of claim 179, where the subjecthas been identified as having CVD.
 181. The method of claim 179, wherethe level of a marker of CVD in the subject has been measured or will bemeasured.
 182. The method of claim 179, where the number of circulatingendothelial cells (CECs) in the blood of the subject has been measuredor will be measured.
 183. The method of claim 182, where the CECs areiNOS-positive circulating endothelial cells.
 184. The method of claim179, where the administering reduces the level of circulatingendothelial cells in the subject.
 185. The method of claim 184, wherethe administering reduces the level of hemoglobin Alc or fasting bloodglucose in the subject.
 186. The method of claim 158, wherein thesubject is a human.
 187. The method of claim 158, wherein at least aportion of the compound is present as a crystalline form having an X-raydiffraction pattern (CuKα) comprising significant diffraction peaks atabout 8.8, 12.9, 13.4, 14.2 and 17.4° 2θ.
 188. The method of claim 187,wherein the X-ray diffraction pattern (CuKα) is substantially as shownin FIG. 12A or FIG. 12B.
 189. The method of claim 187, wherein thepharmaceutically effective amount is a daily dose of about 10 mg toabout 200 mg of the compound.
 190. The method of claim 158, wherein atleast a portion of the compound is present as an amorphous form havingan X-ray diffraction pattern (CuKα) with a halo peak at approximately13.5° 2θ, substantially as shown in FIG. 12C, and a T_(g) from about120° C. to about 135° C.
 191. The method of claim 190, wherein the T_(g)is from about 125° C. to about 130° C.
 192. The method of claim 190,wherein the pharmaceutically effective amount is a daily dose from about0.1 mg to about 30 mg of the compound.
 193. The method of claim 158,where the compound is administered orally, intraarterially orintravenously.
 194. The method of claim 158, where the compound isformulated as a hard or soft capsule or a tablet.
 195. The method ofclaim 158, wherein the compound is formulated as a solid dispersioncomprising (i) the compound and (ii) an excipient.
 196. The method ofclaim 195, wherein the excipient is a methacrylic acid-ethyl acrylatecopolymer (1:1).
 197. A method of improving kidney function in a subjectcomprising administering to the subject a compound of the formula,

in an amount sufficient to improve kidney function, wherein: (a) atleast a portion of the compound is present as an amorphous form havingan X-ray diffraction pattern (CuKα) with a halo peak at approximately13.5° 2θ, substantially as shown in FIG. 12C, and a T_(g) from about120° C. to about 135° C.; and (b) where the subject has been identifiedas having chronic kidney disease (CKD).